Enzymatic cycling assays for homocysteine and cystathionine

ABSTRACT

The present invention provides an enzymatic cycling assay for assessing the amount of homocysteine and/or cystathionine in a solution such as blood, blood derivatives, or urine. The assay comprises the steps of contacting the solution containing homocysteine and/or cystathionine to form a reaction mixture, with CBS, or a derivative thereof, L-serine, and CBL, or a derivative thereof, for a time period sufficient to catalyze the cyclical conversion of homocysteine form to cystathionine and the reconversion of cystathionine to homocysteine with the production of pyruvate and ammonia; determining the amount of homocysteine and/or ammonia present in the reaction mixture; and determining the amount of homocysteine and/or cystathionine present in the solution based on the amount of pyruvate and/or ammonia formed. Expression vectors and isolation procedures for CBS, or derivatives thereof, and CBL, or derivatives thereof, are also provided as well as test kits for carrying out the assay. In preferred embodiments, the assays can be conducted in 15 minutes or less, with a minimum of enzyme usage.

RELATED APPLICATIONS

[0001] This application is a continuation of Ser. No. 10/012,762, filedNov. 6, 2001, which is a continuation-in-part application of U.S. patentapplication Ser. No. 09/704,036, filed Nov. 1, 2000 which claimedbenefit from Provisional Patent Applications Serial No. 60/163,126,filed Nov. 2, 1999 and Serial No. 60/203,349 filed May 10, 2000, and thecontent and teachings of each of these previous applications isincorporated herein by reference.

SEQUENCE LISTING

[0002] A compliant Sequence Listing containing 21 sequences in the formof a computer readable ASCII file in connection with the presentinvention was filed in U.S. Ser. No. 10/012,762 and is incorporatedherein by reference. Applicants request that this earlier-filed CRF beused as the CRF for this application. A paper copy of this sequence isincluded herein and is identical to this previously filed CRF.

BACKGROUND OF THE INVENTION

[0003] High levels of homocysteine in human plasma are correlated withincreased risks for coronary heart disease, stroke, arteriosclerosis,and other diseases. As a result, it is desirable to screen the generalpopulation for elevated amounts of this amino acid. To make wide-scaletesting for homocysteine feasible, new and less expensive assays need tobe developed.

[0004] Plasma homocysteine is routinely measured by high-pressure liquidchroma-tography (HPLC) and gas chromatography/mass spectrometry (GC/MS)at a cost of over $100 per assay, making these physical separationmethods too costly for a population-wide study. For example, urine orblood samples can be prepared for amino acid chromatography, andL-homocysteine measured by HPLC separation and detection. Fiskerstrandet al. (Clin. Chem., 39:263-271 (1993)) describe a method of assayingL-homocysteine using fluorescent labeling of serum thiols, followed byHPLC separation and detection of the L-homocysteine derivative from thevarious other sulfur-containing compounds. However, such methods aretypically time consuming, costly, and not readily available to manylaboratories.

[0005] Indirect immunoassays for homocysteine have also been developed,however, these antibody methods are still relatively expensive at about$24 per test. One particular indirect immunoassay enzymatically convertshomocysteine to adenosyl homocysteine and the amount of adenosylhomocysteine is determined by a competitive ELISA (enzyme linkedimmunoassay), using an anti-adenosyl homocysteine antibody (for example,see U.S. Pat. No. 5,827,645, the content of which is incorporated hereinby reference).

[0006] Indirect enzyme assays have been developed for the quantitationof L-homocysteine. For example, the enzyme S-adenosyl-homocysteinehydrolase and adenosine are added to a test sample. The resultingconcentration, or change in the concentration, of adenosine in thereaction mixture is used as an indicator for the initial concentrationof homocysteine in the sample.

[0007] Direct enzyme assays have also been reported for measuringhomocysteine. Typically, these protocols irreversibly converthomocysteine to other compounds that are quantifiable. For example, theenzyme homocysteine dehydratase has been used to remove the sulfhydrylgroup from homocysteine. The concentration of the removed sulfhydrylmoiety is then measured. A major drawback with this and other enzymeassays for homocysteine is that the enzymes employed react with othersulfur containing amino acids and compounds related to homocysteine,leading to a high and inconsistent background and measurements ofhomocysteine from plasma that are inaccurate.

[0008] Enzymatic (or enzymic) cycling assays have been reported for avery small number of analytes. In an enzymatic cycling assay two or moreenzymes activities are used which recycle substrate and do notirreversibly convert the measured compound. Instead the “compound” isused catalytically to control the rate of conversion to the quantitatedcompound in the assay. As a result, the analyte of interest remains in asteady-state concentration which is low enough to create a pseudo-firstorder rate of reaction. The steady-state concentration of the analyte isthereby linearly related to the rate of the overall assay. By measuringreaction rates, the amount of the analyte is easily determined.Enzymatic cycling assays are sometime called “amplification” assays,because the methods typically increase the sensitivity of measurementfor an analyte by 100- to 1000-fold. The amplification in measurement isa direct result of not reducing the steady-state concentration of thecompound. No enzymatic cycling assay has been reported for measuringhomocysteine.

[0009] The present invention provides an enzymatic cycling assay forhomocysteine and/or cystathionine which is less expensive, and providesa higher sample throughput than the diagnostic assays currentlyavailable. Further, the invention provides methods and vectors for therecombinant production of enzymes which can be used in the production ofassay reagents and test kits for assessing the amount of homocysteineand cystathionine in a sample.

SUMMARY OF THE INVENTION

[0010] The present invention provides an enzymatic cycling assay methodfor assessing the amount of homocysteine and/or cystathionine in asolution. The assay takes advantage of the reaction of homocysteine andL-serine to form cystathionine by the enzyme cystathionine β-synthase(CBS), or a derivative thereof, and the enzymatic conversion bycystathionine β-lyase (CBL) of cystathionine to homocysteine, pyruvateand ammonia. The assay provides a steady-state concentration of thehomocysteine and/or cystathionine which is linearly related to the rateof the overall reaction. The amount of homocysteine and/or cystathioninedetermined in a sample is based on the amount of pyruvate and/or ammoniawhich is formed or the amount of serine removed from the reactionmixture. Solutions which can be tested using the assay of the presentinvention can include laboratory samples of blood, serum, plasma, urine,and other biological samples. Additionally, any other liquid sample canbe tested.

[0011] In one embodiment, the present invention provides a method forassessing the amount of homocysteine and/or cystathionine in a solutioncomprising the step of:

[0012] (a) contacting the solution containing homocysteine and/orcystathionine (either before and/or after performing a disulfidereduction step) to form a reaction mixture, with CBS, or a derivativethereof, L-serine and CBL, or a derivative thereof, for a time periodsufficient to catalyze the cyclical conversion of homocysteine tocystathionine, and the reconversion of cystathionine to homocysteinewith the production of pyruvate and ammonia;

[0013] (b) determining the amount of pyruvate and/or ammonia present inthe reaction mixture; and

[0014] (c) assessing the amount of homocysteine and/or cystathioninepresent in the solution based on the amount of pyruvate and/or ammoniaformed.

[0015] More particularly, the method provides for the assessment of theamount of homocysteine by the addition of the inexpensive amino acidL-serine. The amount of pyruvate present in the reaction mixture can bemeasured in a number of ways. In one particular embodiment of thepresent invention lactate dehydrogenase (LDH), or a derivative thereof,and NADH (reduced nicotinamide cofactor), or a derivative thereof, arepresent in the reaction mixture. LDH in the presence of NADH convertspyruvate to lactate with the oxidation of NADH to NAD+(oxidizednicotinamide cofactor).

[0016] The oxidation of NADH to NAD+can be measured by a number ofmethods known in the art including monitoring the reaction mixture at340 nm. Production of NAD+can also be monitored by the oxidation of adye to produce an observable color change. Dyes preferred for use in thepresent invention include, but are not limited to,5,5′-dithiobis(2-nitrobenzoic acid), 2,6-dichlorophenylindophenol,tetrazolium compounds, phenazine methosulfate, methyl viologen, orderivatives of any of these dyes. The amount of homocysteine and/orcystathionine present in the solution is based on the intensity of theobserved color compared to a standard curve constructed for samples ofknown concentration of the analyte.

[0017] In an alternative embodiment pyruvate oxidase, with horseradishperoxidase, in the presence of hydrogen peroxide and a chromogen areused to detect the amount of pyruvate present in the sample. SodiumN-ethyl-N-2-hydroxy 3-sulfopropyl) m-toluidine (TOOS) and otherN-ethyl-N-(2-hydroxy-3-sulfopropyl)-aniline derivatives are preferredchromogens for this colorimetric reaction. As above, the amount ofhomocysteine and/or cystathionine present in the sample is based on theintensity of the observed color compared to a standard curve constructedfor samples of known concentrations of the analyte.

[0018] The amount of homocysteine and/or cystathionine present in asolution can also be measured based on the amount of ammonia present inthe reaction mixture. Methods for determining the concentration ofammonia in a solution are legion. In one particular embodiment of thepresent invention, the amount of ammonia is measured using a commonlyavailable standard ammonia sensor.

[0019] In another embodiment, the present invention is directed to amethod for assessing the amount of homocysteine and/or cystathionine ina sample comprising the steps of contacting the solution with a reducingagent for a time period sufficient to reduce substantially allhomocysteine and other disulfides that are present in the solution tohomocysteine. Treatment with a reducing agent can also act to releasehomocysteine which is attached to a protein and/or other moleculespresent in a solution through a disulfide bond. After reduction thesolution is then contacted with CBS, or a derivative thereof, L-serine,and CBL, or a derivative thereof, for a time period sufficient tocatalyze the cyclical conversion of homocysteine to cystathionine andthe conversion of cystathionine to homocysteine with the production ofpyruvate and ammonia. To assess the amount of homocysteine and/orcystathionine present in the solution the amount of pyruvate and/orammonia present in the reaction mixture can be determined as set forthabove. Preferred reducing agents for use in the present inventioninclude borohydride salts and thiol reducing agents. Typical thiolreducing agents appropriate for use in the present embodiment includedithioerythritol (DTE), dithiothreitol (DTT), β-mercaptoethanol (βME),tris(carboxyethyl)phosphine hydrochloride (TCEP), or thioacetic acid, orany derivatives thereof, and the like.

[0020] In yet another embodiment for assessing the amount ofhomocysteine and/or cystathionine present in a solution, the solutioncan be pretreated with cystathionine γ-lyase (CGL), or a derivativethereof, for a time period sufficient to remove any cystathionine fromthe reaction mixture by the conversion of cystathionine toα-ketoglutarate. Following cystathionine removal the cystathionineγ-lyase is removed from the reaction mixture or destroyed. In a typicalembodiment, the cystathionine γ-lyase is destroyed by heating thesolution for a time period sufficient to remove substantially itsenzymatic activity. The cystathionine γ-lyase can also be immobilized onan insoluble substrate or surface, such as, for example, a microparticle or bead, which can be easily removed.

[0021] In still another embodiment of the present invention a method isprovided for assessing the amount of homocysteine and/or cystathioninepresent in a solution comprising the reaction of the solution withL-serine and CBS, or a derivative thereof, and CBL, or a derivativethereof, which have been immobilized on a solid surface. The solidsurface can be, for example, paper, filter paper, nylon, glass, ceramic,silica, alumina, diatomaceous earth, cellulose, polymethacrylate,polypropylene, polystyrene, polyethylene, polyvinylchloride, andderivatives thereof. The solid surface can be the sides and bottom ofthe test container or can be a material added to the test container. Ina preferred embodiment the solid surface comprises a bead which is addedto the test container.

[0022] The CBS, or derivative thereof, CBL, or derivative thereof, andcystathionine γ-lyase, or derivative thereof, useful in the presentinvention can be obtained as a crude extract from a cell. In oneembodiment of the present invention the cystathionine β-synthase (CBS),or derivative thereof, cystathionine β-lyase CBL, and/or cystathionineγ-lyase (CGL) are purified from human, yeast or bacterial cells. In aparticularly preferred embodiment of the present invention the geneswhich encode the enzymes are isolated or synthesized and are expressedas a recombinant protein in a host cell. It is particularly preferredthat a DNA sequence which encodes an affinity tag be added to the geneconstruct to aid in the purification and/or detection of therecombinantly produced enzymes. Recombinant methods can also be used toprovide fusion proteins which comprise the enzyme activities of CBS andCBL in a single protein. An affinity tag can also be included as part ofthe fusion protein construct to aid in the purification of the fusionprotein.

[0023] The present invention also provides as a method for assessing theamount of homocysteine in a sample an assay format which correlates theamount of homocysteine/transcription factor complex which is bound to aconsensus polynucleotide binding sequence. In a particular embodimentthe method comprises contacting the sample with a reducing agent for atime period sufficient to release homocysteine from any associatedprotein; contacting the reduced homocysteine with a homocysteinemetabolite binding transcription factor under conditions conducive forcomplex formation, admixing the sample with a consensus polynucleotidesequence specifically recognized by the homocysteine/transcriptionfactor complex; and assessing from the amount ofhomocysteine/transcription factor complex bound to the consensuspolynucleotide sequence the amount of homocysteine present in thesample. Reducing agents which are applicable for use in the methodcomprise borohydride salt or thiol reducing agents includingdithioerythritol (DTE), dithiothreitol (DTT), β-mercaptoethanol,tris(carboxyethyl)phosphine (TCEP), or thioacetic acid, or any salt ofeach. Homocysteine metabolite binding transcription factors include MetRof E. coli which recognizes a consensus polynucleotide sequence, forexample, the polynucleotide sequence as depicted in SEQ ID NO: 11(Marconi et al., Biochem. Biophys. Res. Commun., 175:1057-1063 (1991))or a derivative thereof.

[0024] Yet another embodiment of the present invention provides a testkit comprising a container for holding the solution to be assessed forthe amount of homocysteine and/or cystathionine, L-serine, CBS, or aderivative thereof, CBL, or a derivative thereof, and any buffers,auxiliary substances and solvents required to provide conditionsconducive to high enzyme activity. The test kit can further compriselactate dehydrogenase, or a derivative thereof, and NADH, or aderivative thereof. NADH can be measured directly at 340 nm or, a dyecapable of providing a color change when oxidized can be included. Thequantity of homocysteine and/or cystathionine is correlated with thechange in absorbance measure over time.

[0025] In a preferred embodiment the enzymes are provided immobilized toa solid support. The solid support can comprise the surface of thecontainer provided to hold the test sample or can be a bead or otherarticle added to the container. In an additional embodiment of thepresent invention, cystathionine γ-lyase can be provided as part of thetest kit to remove any cystathionine from the test solution prior to theenzymatic cycling assay. Substantially all of the activity of thecystathionine γ-lyase, or derivative thereof, must be removed from ordestroyed in the reaction mixture prior to the addition of the remainingcomponents for the enzymatic cycling of homocysteine.

[0026] It has been found that the preferred assay of the invention canbe carried out in a relatively short period of time and with relativelysmall amounts of enzyme, giving an assay which has substantialcommercial advantages. For example, the preferred assay involvescreation of a reaction mixture including a homocysteine-containingsample, serine, CBS, CBL, lactate dehydrogenase, NADH and a reductantsuch as DTE or DTT, with the CBS/CBL ratio in the mixture being fromabout 1:1 to 25:1, more preferably from about 1:10, and most preferablyfrom about 2:1 to 5:1. Advantageously, it has been found that thereductant can be present along with the detection system (i.e., thelactate dehydrogenase and NADH) without deleteriously affecting theassay. Accordingly, the assay of the invention can be carriedout-without a separate reduction step so that total assay times arereduced. Thus, where a plurality of samples are to be assayed bysequentially creating a reaction mixture in a container (e.g., aspectrophotometric cuvette) made up of a sample, serine, CBS, CBL,lactate dehydrogenase, NADH and the reductant, and assessing the amountof pyruvate present in the reaction mixture by monitoring the productionof NAD+ over time, the time interval between the respectivereaction-creation steps may be as short as 10-50 seconds with the totalreaction time being up to about 20 minutes for a given sample. Morepreferably, the total reaction time for a given sample is up to about 15minutes, and still more preferably up to about 13 minutes. The totalnumber of samples assayed per hour may be about 15-30 for slowerinstruments, but as high as 200-400 for faster instruments. In thispreferred assay, a first reaction mixture comprising the sample, serine,lactate dehydrogenase, NADH and the reductant is prepared, with asuitable incubation period to permit liberation of a preponderance (andpreferably essentially all) of the homocysteine (total homocysteine)present in the bound, oxidized and/or free states in the sample.Thereafter, CBS and CBL are added to complete the reaction mixture andinitiate enzymatic cycling of homocysteine and/or cystathionine.

[0027] In another preferred embodiment, cystathionine can be used tomake a calibrator(s) for the enzyme assay (since the enzymatic cyclingassay interconverts homocysteine and cystathionine). In other words,varying known levels of cystathionine can be used in the assay system to“calibrate” or “standardize” the assay and/or instrument which allowsfor quantitation of sample results. In this embodiment, a knownconcentration of cystathionine is added to a biological sample and thensubjected to the assay used for one of the other embodiments. Theresults will be used to establish a calibration line which will then beused to set the homocysteine line, due to the high degree of correlationbetween the two lines. Alternatively, known levels of cystathionine canbe used as a quality control measure, to insure that the assay isworking properly. In this quality control embodiment, these “known”levels of cystathionine are assayed as if they were unknown samples, andthe results are compared to their known (expected) values, in order toinsure that the assay system is functioning properly.

[0028] The preferred assays are carried out using isolated (purified)CBL and CBS enzymes having at least about 80% (and preferably at leastabout 90%) sequence identity with the enzymes selected from the groupconsisting of SEQ ID Nos. 19 and 20. As used herein, “sequence identity”as it is known in the art refers to a relationship between two or moreprotein or polypeptide sequences or two or more polynucleotidesequences, namely a reference sequence and a given sequence to becompared with the reference sequence. Sequence identity is determined bycomparing the given sequence to the reference sequence after thesequences have been optimally aligned to produce the highest degree ofsequence similarity, as determined by the match between strings of suchsequences. Upon such alignment, sequence identity is ascertained on aposition-by-position basis, e.g., the sequences are “identical” at aparticular position if at that position, the nucleotides or amino acidresidues are identical. The total number of such position identities isthen divided by the total number of nucleotides or residues in thereference sequence to give % sequence identity. Sequence identity can bereadily calculated by known methods, including but not limited to, thosedescribed in Computational Molecular Biology, Lesk, A. N., ed., OxfordUniversity Press, New York (1988), Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York (1993); ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey (1994); Sequence Analysis in MolecularBiology, von Heinge, G., Academic Press (1987); Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York(1991); and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073(1988), the teachings of which are incorporated herein by reference.Preferred methods to determine the sequence identity are designed togive the largest match between the sequences tested. Methods todetermine sequence identity are codified in publicly available computerprograms which determine sequence identity between given sequences.Examples of such programs include, but are not limited to, the GCGprogram package (Devereux, J., et al., Nucleic Acids Research, 12(1):387(1984)), BLASTP, BLASTN and FASTA (Altschul, S. F. et al., J. Molec.Biol. 215:403-410(1990). The BLASTX program is publicly available fromNCBI and other sources (BLAST Manual, Altschul, S. et al., NCVI NLM NIHBethesda, Md. 20894, Altschul, S. F. et al., J. Molec. Biol.,215:403-410 (1990), the teachings of which are incorporated herein byreference). These programs optimally align sequences using default gapweights in order to produce the highest level of sequence identitybetween the given and reference sequences. As an illustration, by apolynucleotide having a nucleotide sequence having at least, forexample, 95% “sequence identity” to a reference nucleotide sequence, itis intended that the nucleotide sequence of the given polynucleotide isidentical to the reference sequence except that the given polynucleotidesequence may include up to 5 point mutations per each 100 nucleotides ofthe reference nucleotide sequence. In other words, in a polynucleotidehaving a nucleotide sequence having at least 95% identity relative tothe reference nucleotide sequence, up to 5% of the nucleotides in thereference sequence may be deleted or substituted with anothernucleotide, or a number of nucleotides up to 5% of the total nucleotidesin the reference sequence may be inserted into the reference sequence.These mutations of the reference sequence may occur at the 5′ or 3′terminal positions of the reference nucleotide sequence or anywherebetween those terminal positions, interspersed either individually amongnucleotides in the reference sequence or in one or more contiguousgroups within the reference sequence. Analogously, by a polypeptidehaving a given amino acid sequence having at least, for example, 95%sequence identity to a reference amino acid sequence, it is intendedthat the given amino acid sequence of the polypeptide is identical tothe reference sequence except that the given polypeptide sequence mayinclude up to 5 amino acid alterations per each 100 amino acids of thereference amino acid sequence. In other words, to obtain a givenpolypeptide sequence having at least 95% sequence identity with areference amino acid sequence, up to 5% of the amino acid residues inthe reference sequence may be deleted or substituted with another aminoacid, or a number of amino acids up to 5% of the total number of aminoacid residues in the reference sequence may be inserted into thereference sequence. These alterations of the reference sequence mayoccur at the amino or the carboxy terminal positions of the referenceamino acid sequence or anywhere between those terminal positions,interspersed either individually among residues in the referencesequence or in the one or more contiguous groups within the referencesequence. Preferably, residue positions which are not identical differby conservative amino acid substitutions. However, conservativesubstitutions are not included as a match when determining sequenceidentity.

[0029] Reagents useful in the present invention include CBS, CBL,L-serine, LDH, NADH, DTE, βME, DTT, TCEP, thioacetic acid, and CGL.Concentrations of CBS enzyme useful in the present invention range fromabout 0.1 to about 100 KU/I. More preferably, the CBS concentration isfrom about 0.5 to about 75 KU/1, and still more preferably from about 1to about 50 KU/1. Most preferably, the concentration of CBS is fromabout 1 to about 30 KU/I. Concentrations of CBL enzyme useful in thepresent invention range from about 0.01 to about 100 KU/1. Morepreferably, CBS concentration is from about 0.05 to about 50 KU/I, andstill more preferably, from about 0.1 to about 30 KU/1. Most preferably,the CBS concentration is from about 0.1 to about 15 KU/1. L-serine maybe present at a final concentration of from about 1 μM to about 50 mM.More preferably, the L-serine is added at a final concentration of fromabout 10 μM to about 40 mM, and still more preferably at a finalconcentration of from about 100 μM to about 20 mM. Most preferably, thefinal concentration of L-serine added is from about 0.2 mM to about 10mM. When used in any embodiment, LDH is present in the reaction mixtureat a final concentration of from about 30 to about 5000 U/L. Morepreferably, the final concentration of the LDH present in the reactionmixture is from about 30 to about 3000 U/L, and still more preferablyfrom about 50 to about 2500 U/L. Most preferably, LDH is present in thereaction mixture at a final concentration of from about 100 to about2000 U/L. Additionally, when NADH is used in any embodiment, the amountof NADH present in the reaction mixture can vary between about 0.1 mM toabout 2 mM. More preferably, NADH is present in the reaction mixture ata final concentration of from about 0.1 mM to about 1.5 mM, and stillmore preferably between about 0.1 to about 1 mM. Most preferably, NADHis present in the reaction mixture at a final concentration of fromabout 0.2 to about 0.8 mM. When DTE is used in the present invention, itcan range in final concentration from about 0.01 mM to about 100 mM.More preferably, concentrations of DTE will range from about 0.01 mM toabout 50 mM, and still more preferably from about 0.1 mM to about 25 mM.Most preferably, final concentrations of DTE will range from about 0.1mM to about 10 mM. Similarly, when DTT is used in the present invention,it can range in final concentration from about 0.01 mM to about 100 mM.More preferably, concentrations of DTT will range from about 0.01 mM toabout 50 mM, and still more preferably from about 0.1 mM to about 25 mM.Most preferably, final concentrations of DTT will range from about 0.1mM to about 10 mM. Finally, when CGL is used in the present invention,it can range in final concentration from about 0.1 KU/I to about 100KU/I. More preferably, CGL will range from about 0.5 KU/I to about 75KU/I, and still more preferably from about 1 KU/I to about 50 KU/I. Mostpreferably, final concentrations of CGL will range from about 1 KU/I toabout 30 KU/I.

[0030] In some preferred embodiments, the buffer of Reagent 1 is TRIS,250 mM at pH 8.4. However, when TRIS is used as the buffer the pH mayrange from 7.0-9.0. More preferably, the pH ranges from 7.5-8.5 andstill more preferably from about 8.1-8.5. The use of increasedconcentration of TRIS improves assay consistency by helping to maintaina more constant pH as it is a more concentrated buffer. Additionally,CBS and CBL are substantially more active in this preferred pH range,and especially from about pH 8.1-8.5.

[0031] The present invention also provides an assay which works equallywell with turbid samples. Problems with turbidity are reduced by addinglipase and-cyclodextrin to R1. These help to decrease turbidity by thehydrolysis of triglycerides to glycerol and free fatty acids by lipaseand by the formation of complexes with the free fatty acids by theα-cyclodextrin. The addition of these two components helped to clear thereaction mixture before the addition of Reagent 3. Replacing lipase andα-cyclodextrin with EDTA provides another means of accurately analyzingturbid samples.

[0032] Another variation of the present invention tested the effects ofvarying volumes of the reagents. Such variations did not have asubstantial effect on the accuracy of the assay.

[0033] The present invention also tested the effects of differentdetergents and concentrations thereof. For example, Genapol X-80 may bepresent in Reagent 1 at a concentration between about 0.05-0.5%. Morepreferably, the concentration is between 0.1-0.5% and still morepreferably between about 0.2-0.4%. Of these, three concentrations (0.1%,0.3%, and 0.5%) were tested with the 0.3% concentration providing themost accurate results. Brij-35 was also varied in concentration inReagent 1. Brij-35 may be used with the present invention in R1 at aconcentration between about 0.01-0.5%. More preferably, thisconcentration ranges from about 0.015-0.1%, and still more preferablyfrom about 0.020-0.030%. Of the concentrations tested (0.025%, 0.05%,0.1% and 0.5%), the most accurate and consistent results were obtainedusing a concentration of 0.025%.

[0034] The present invention also tested replacing the reducing agentDTE with Tris (2-carboxyethylphosphine) hydrochloride (TCEP) which isstable in purified water for an extended period of time. Such asubstitution reduced the reagent blank reaction from about 12-15 mA/minto about 4-5 mA/min which enables a greater degree of precision for thisassay. When TCEP is used in the present invention, it may range inconcentration from about 6-53 mM. More preferably, this concentrationranges from about 10-45 mM and still more preferably from about 20-30mM. In the concentrations tested in this application, 26.44 mM providedthe most accurate assay results.

[0035] The mixture comprising Reagent 3 was also varied in composition.For example, the Tris buffer was replaced with a phosphate buffer andglycerol was added to the buffer. The phosphate was effective atconcentrations ranging from about 50 mM to about 500 mM at pH 7.6. Theglycerol concentrations ranged from about 0.5% to 15.0% (v/v). Phosphateand glycerol were used to provide longer shelf-life for the enzymes,which was not possible with the TRIS buffer.

[0036] One of the advantages of the present invention is that it may beused with any of a number of instruments. For example, the assay wasadapted for testing on two different instruments, the Hitachi 911 andthe Beckman CX5. Notably, accurate and consistent results were obtainedwith either machine. Additionally, the assay can be run using just 2reagents by mixing the components of R1 and R2 together in the correctratio based on the instrument requirements.

[0037] Finally, the present invention can be adapted for use with asingle calibration point. Importantly, the results from single pointcalibration was just as accurate as a multi-point calibration curve.

BRIEF DESCRIPTION OF THE DRAWINGS

[0038]FIG. 1 depicts absorbance versus time plots obtained using aqueoussolutions of pyruvate (5-100 μM) demonstrating the quantitation ofpyruvate. Reagent components were those described in Table 1. andinstrument settings those described in Table 2;

[0039]FIG. 2 depicts a standard curve of absorbance values plottedversus known quantities of pyruvate (0-100 μM). Assay time was 5 min.;

[0040]FIG. 3 depicts absorbance versus time plots obtained using aqueoussolutions of cystathionine (0-100 μM). Cystathionine was measured by theenzymic conversion of cystathionine to homocysteine, pyruvate andammonia by CBL;

[0041]FIG. 4 depicts cystathionine assay standard curves obtained usingaqueous solutions of cystathionine (0 to 100 μM). Reaction time waseither 5 or 20 min.;

[0042]FIG. 5 depicts absorbance versus time plots demonstrating theCBS/CBL/pyruvate oxidase/peroxidase cycling and signaling system.Cystathionine was dissolved in water to prepare standards atconcentrations between 1 and 100 μM;

[0043]FIG. 6 depicts absorbance versus time plots demonstrating theCBS/CBL/pyruvate oxidase/peroxidase cycling and signaling system.Homocysteine was dissolved in water to prepare standards atconcentrations between 0 and 100 μM;

[0044]FIG. 7 depicts a typical calibration curve obtained using aqueoussolutions of homocysteine (0-100 μM) using the CBS/CBL/PO/HRP cyclingand detection system;

[0045]FIG. 8 depicts typical absorption versus time plots obtained usinghomocysteine (0-100 μM) in aqueous solution using the CBS/CBL/LDH systemwithout dithiotreitol;

[0046]FIG. 9 depicts typical absorption versus time plots obtained usinghomocysteine (0-100 μM) in aqueous solution using the CBS/CBL/LDHcycling system with dithiotreitol;

[0047]FIG. 10 depicts a calibration plot of homocysteine concentration(0 to 100 μM) versus absorbance with and without dithiotreitol. Reactiontime was 5 minutes;

[0048]FIG. 11 depicts a correlation between the quantitation resultsobtained using the CBS/CBL/LDH enzyme cycling system to measurehomocysteine in human plasma samples using DTT as the reducing agentcompared with quantitation results obtained by the Abbott IMxhomocysteine method;

[0049]FIG. 12 depicts a correlation graph illustrating the correlationbetween the assay of the present invention (y) versus the commerciallyavailable IMx assay (x), as described in Example 7;

[0050]FIG. 13 depicts a correlation graph illustrating the correlationbetween the assay of the present invention (y) versus a conventionalHPLC assay (x), as described in Example 7;

[0051]FIG. 14 depicts a correlation graph illustrating the correlationbetween the IMx assay (x) and the HPLC assay (y), as described inExample 7;

[0052]FIG. 15 depicts a graph of absorbance versus time for the no-DTTpyruvate assay described in Example 9; and

[0053]FIG. 16 depicts a graph of absorbance versus time for theDTT-containing pyruvate assay described in Example 9.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

[0054] The present invention provides a homogeneous enzymatic cyclingassay for assessing the amount of homocysteine in a sample. Enzymaticcycling maintains a steady-state level of homocysteine and furtherprovides an increase in the sensitivity of assaying homocysteine.Further, the present invention provides genes and expression vectors forthe recombinant production of enzymes that are useful in the presentinvention. Test kits for assessing the amount of homocysteine and/orcystathionine in a solution are also provided.

[0055] Homocysteine is present in cells as an intermediary amino acidproduced when methionine is metabolized to cysteine. Generally,homocysteine produced in the body is rapidly metabolized by one of tworoutes: (1) condensation with serine to form cystathionine or (2)conversion to methionine, and its concentration (and that of itsoxidized form, homocystine) in the body under normal conditions is low.The average plasma homocysteine level for a healthy adult is about 12 μMfor males ranging from about 8.6 to about 17.1 μM and 10 μM for femalesranging from about 3.9 to 16.8 μM (Vester et al., Eur. J. Clin. Chem.Biochem., 29:549-554 (1991); Jacobsen et al. Clin. Chem., 40:873-881(1994)).

[0056] Recently it has been determined that the amount of homocysteinelevels in a biological sample is of clinical significance in a number ofconditions. Elevated amounts of homocysteine in the blood have beencorrelated with the development of atherosclerosis (Clarke et al., NewEng. J. Med., 324:1149-1155 (1991)). Also, moderate homocysteinemia isnow regarded as a risk factor for cardiac and vascular disease. Accuracyin the normal range of homocysteine found in normal adults is veryimportant since the risk for heart disease has been determined toincrease markedly with as little as 30% over the normal concentrationincreasing the associated risk factor by 3.4 fold (Stampfer et al., JAMA268:877-881 (1992)).

[0057] As used herein the term “assessing” is intended to include bothquantitative and qualitative determination of the amount of homocysteineand/or cystathionine present in a test solution. A “solution” or “testsolution” as used herein refers to a clinical or biological fluid sampleor tissue extract and can include, for example, blood, bloodderivatives, such as plasma, or urine, and the like.

[0058] A sample containing homocysteine reacts with L-serine to formcystathionine in the presence of the enzyme CBS. Cystathionine can thenbe converted to homocysteine, pyruvate, and ammonia with a secondenzyme, CBL. The result of using these anabolic and catabolic enzymesoperating simultaneously is a “futile cycle,” whereby (1) cystathionineand homocysteine are continuously interconverted, (2) serine isdegraded, and (3) pyruvate and ammonia are created.

[0059] Use of relatively high concentrations of serine compared tohomocysteine provides for conditions conducive to an overall reactionrate which follows pseudo-first order kinetics dependent directly uponthe amount of homocysteine and cystathionine in the reaction. If theconcentration of cystathionine is very low compared to the concentrationof homocysteine, the rate of the overall reaction will vary linearlywith the amount of homocysteine. By measuring the reduction in serine orthe increase in either pyruvate or ammonia, the amount of homocysteinein an unknown solution or sample can be determined by a comparison withcontrol reactions comprising known concentrations of homocysteineundergoing the identical enzyme reactions. The enzymatic cycling methodof the present invention provides an amplification process producing fargreater amounts of end products than the amount of homocysteine likelyto be in a test solution, i.e., blood. Therefore, the assays todetermine the amount of pyruvate or ammonia produced by the reaction donot have to be extremely sensitive and can be measured by existingprocesses known to the skilled artisan.

[0060] Pyruvate is typically measured by the enzymatic conversion intolactate with lactate dehydrogenase, or a derivative thereof. Thisenzymatic conversion reaction requires the cofactor NADH (reducednicotinamide cofactor), or a derivative thereof, which is converted toNAD+(oxidized nicotinamide cofactor). The reaction can be monitored bymeasuring the absorbance at 340 nm. As used herein, the term“derivatives,” with respect to cofactors, refers to salts, solvates, andother chemically modified forms that retain overall cofactor activity,but can have extended stability in aqueous solutions. Derivatives caninclude, but are not limited to acetyl derivatives of NADH, including3-pyridinealdehyde-NADH, 3-acetylpyridine-NADH, 3-thioicotinamide-NADH,and the like (See, for example, U.S. Pat. No. 5,801,006).

[0061] Other convenient assays to determine the oxidation of NADH, or aderivative thereof, include for example, the measurement offluorescence, as described by Passoneau et al., in Enzymatic Analysis, APractical Guide, pages 219-222 (1993), (incorporated herein byreference). In another embodiment, NADH, or a derivative thereof, reactswith pyruvate in the presence of lactate dehydrogenase to form NAD+,which further reacts with a dye capable of producing a color change,preferably in the visible range, when oxidized. The color change of thedye can be used to determine the total amount of oxidation of NADH toNAD+ which corresponds to the amount of pyruvate formed from theenzymatic cycling reaction. Examples of dyes which can be used in thepresent invention include, but are not limited to,5,5′-dithiobis(2-nitrobenzoic acid), 2,6-dichlorophenolindophenol,tetrazolium compounds, phenazine methosulfate, methyl viologen, andderivatives of each.

[0062] Pyruvate can also be measured in a reaction with pyruvateoxidase, or a derivative thereof, in the presence of a peroxidase, i.e.,horseradish peroxidase, and the like. The amount of pyruvate convertedto hydrogen peroxide is determined by the oxidative condensation of, forexample, a water soluble hydrogen donor which provides a coloredcompound for photometric determination of the concentration of peroxide.Particularly preferred hydrogen donors which provide a stable colorreaction product include water soluble derivatives ofN-alkyl-N-sulfopropylaniline derivatives, i.e., the sodium salt ofN-ethyl-N-(2-hydroxy-3-sulfopropyl)-m-toluidine (TOOS) (Tamaoku et al.,Chem. Pharm. Bull. 30:2492-2497 (1982)). The quantity of H₂O₂ can alsobe measured electrometrically. This includes amperometric andpotentiometric measurements. The determination of the amount ofhomocysteine and/or cystathionine is determined from the correlation ofthe amount of peroxide produced from the conversion of pyruvate bypyruvate oxidase over a defined time period.

[0063] Ammonia also can be measured by different methods, including byuse of an ammonia sensor (Willems et al., Clin. Chem. 34:2372 (1988).Ammonia can also be readily detected using known colorimetrictechniques. For example, ammonia generated in the sample can be reactedto form a colored product, the formation of which can be detectedspectrophotometrically. One such method, described in Methods ofEnzymatic Analysis (Bergmeyer) 1:1049-1056 (1970), incorporated hereinby reference, relies upon the reaction of ammonia with phenol in thepresence of hypochlorite in alkaline conditions to form the colored dyeindophenol. Sodium nitroprusside can be used as a catalyst.Modifications of the method using for example various derivatives canalso be used. The colored end product is formed in amounts directlyproportional to the concentration of ammonia, and hence homocysteine.Other methods can include, for example, microdiffusion analysis(Seligson et al., J. Lab. Clin. Med., 49:962-974 (1957), an ion exchangetechnique (Forman, Clin. Chem., 10:497-508 (1964), and various enzymaticmethods (See, for example, Mondzac et al., J. Lab. Clin. Med.,66:526-531 (1979)).

[0064] The enzymatic cycling method for assessing the amount ofhomocysteine and/or cystathionine reduces the difficulties seen in otherprior assays where background compounds and background reactionsproduced “noise.” In the assay of the present invention, large amountsof an inexpensive amino acid, e.g., L-serine, can be added to providefor the overall conversion of L-serine to pyruvate and ammonia. In apreferred embodiment, a serine concentration of 250 μM or more can beused, and subsequently converted to pyruvate and ammonia. Plasma,typically contains about 100 μM serine. This amount of endogenous serineis immaterial to the present assay since the assay measures a rate ofconversion that is limited by the amount of homocysteine and not theamount of serine. L-serine concentrations of about 0.1 mM to 10 mM wouldnot be expected to affect the sensitivity or accuracy of the disclosedassay.

[0065] Further, the enzymatic cycling reaction of the present inventionprovides a rate of L-serine conversion that is dependent upon theconcentration of homocysteine in a pseudo-first order reaction. Normalblood pyruvate levels are typically between about 0.4 and 2.5 mg/100 ml(Lehninger, A L. Principles of Biochemistry. 1982. WorthingtonPublishers, Inc. p. 707, incorporated herein by reference), whichcorresponds to about 45-284 μM. This concentration of pyruvate couldprovide a minor background measurement, but the present assay measures achange over time and can be easily adjusted for the starting level ofpyruvate in a solution. In addition, pyruvate may be removed by priorincubation with lactate dehydrogenase or other enzymes. Moreimportantly, cysteine and other sulfur-bearing compounds in, forexample, blood, do not contribute significantly to the enzymatic cyclingreaction and, therefore, are considered insignificant noise in thesystem.

[0066] In an alternative embodiment of the present invention the samplecan be treated to remove cystathionine to increase the accuracy of thehomocysteine measurement. As a particular example, treatment comprisescontacting cystathionine γ-lyase with the solution to convertcystathionine to α-ketoglutarate (a compound which can not participatein the enzymatic cycling described above). Prior to proceeding with theenzyme cycling assay to assess the amount of homocysteine, thecystathionine γ-lyase activity must be removed from the sample ordestroyed, i.e., by heat, or the like.

[0067] This particular embodiment of the invention can also be used forthe assessment of the amount of cystathionine by comparing thedifferences in the rate of production of pyruvate and/or ammonia in asample treated and untreated with cystathionine γ-lyase prior toundergoing the enzymatic cycling assay. Therefore, the present inventioncan be used to determine the amount of cystathionine in an unknownsample, even if the sample contains homocysteine.

[0068] In certain biological samples, such as blood, homocysteine isoften bound through a disulfide linkage to proteins, thiols, and othercellular components. Typically, the sample can be treated with areducing agent, such as sodium or potassium borohydride; a thiol, suchas dithioerythritol, β-mercaptoethanol, dithiothreitol, thioglycolicacid or glutathione; or a phosphine or a trialkylphosphine, such astributylphosphine and tris(2-carboxyethyl)phosphine (TCEP), and thelike, to liberate bound and disulfide linked homocysteine. Prior assayswhich require antibodies for the detection step must change the redoxconditions of the reaction mixture (most antibodies are held togetherwith a disulfide bond). However, in the present method it is notnecessary to wash or alter redox conditions because the CBS and CBL usedare active under reducing conditions. Therefore, fewer steps arerequired for the assays of the present invention than for many prior artimmunoassays.

[0069] In a typical embodiment, a sample comprising blood, bloodfractions, serum, plasma, or urine will be treated with a reducing agentand directly assayed for homocysteine. Heating of the sample prior tothe assay to speed up the liberation of homocysteine and to inactivatedegradative enzymes and other factors that may interfere with the assaymay also be preferred. The general handling of blood samples forhomocysteine assays is known to those skilled in the art for othermethods such as HPLC tests.

[0070] CBS and CBL are enzymes commonly found in nature. Humans andyeast use CBS for the synthesis of methionine; and the human cDNA forCBS can substitute for the CBS gene in brewer's yeast (Kruger et al.,Hum. Molec. Genet., 4:1155-1161 (1995)). CBL is found in many bacteria,including E. coli (Laber et al., FEBS Lett., 379:94-96 (1996). Theentire DNA sequences for the CBS and CBL genes are known in theseorganisms and in other species, providing a skilled molecular biologistwith a source to easily clone or synthesize these genes, or derivativesthereof.

[0071] As used herein “derivative” with respect to enzymes refers tomutants produced by amino acid addition, deletion, replacement, and/ormodification; mutants produced by recombinant and/or DNA shuffling; andsalts, solvates, and other chemically modified forms of the enzyme thatretain enzymatic activity of the isolated native enzyme. A method forcreating a derivative enzyme by DNA shuffling useful in the presentinvention is described in U.S. Pat. No. 5,605,793 (incorporated hereinby reference). Even without cloning, the two enzymes have been purifiedfrom a number of different organisms. See, for example, Ono et al.,Yeast 10:333-339 (1994); Dwivedi et al., Biochemistry 21:3064-3069(1982), incorporated herein by reference. In a preferred embodiment ofthe present invention genetically modified organisms are provided toproduce a CBS, or a derivative thereof, and to produce a CBL, or aderivative thereof, which can be used in the enzymatic cycling assay toprovide reagents at a reduced cost and to increase the ease of purifyingthe enzymes.

[0072] Similarly, the term “derivative” with respect to nucleotidesequences refers to variants and mutants produced by nuclueotideaddition, deletion, replacement, and/or modification, and/orcombinatorial chemistry; mutants produced by recombinant and/or DNAshuffling; and salts, solvates, and other chemically modified forms ofthe sequence that retains the desired activity of the sequence such asthe binding characteristics of MetR. Preferably, when modifiedderivative sequences are used, the sequences have at least about 60%sequence identity with the original sequence. More preferably, thisidentity is at least about 70% and more preferably at least about 85%.Most preferably, such sequences have at least about 95% sequenceidentity with the original sequence.

[0073] The CBS gene and the entire genome of Saccharomyces cerevisiaehave been sequenced. Likewise the CBL gene and the remaining genes of E.coli are also known. Therefore, using techniques common to the art ofmolecular biology, the genes encoding CBS and CBL can be cloned andexpressed in both organisms. Published methods for purifying the twoenzymes are known which use standard protein purification methods.However each of the enzymes can be constructed as fusion proteinsincluding for example, a portion of another protein which assists insolubilization and/or refolding of the active enzyme or amino acidsequences which can be added to aid in purification. An example of aminoacid sequences added for purification include, affinity tags, such aspoly-His, or glutathione-S-transferase (GST) can be added to allow forthe more rapid purification of the proteins over a single affinitycolumn. Although the enzymes do not require purification to homogeneityfor a diagnostic test of the present invention, proteases and otheractivities that convert serine to pyruvate can be reduced toinsignificant levels by purification.

[0074] In addition, concentration of the enzymes generally leads tohigher stability of enzyme activity during the reaction and upon longterm storage. CBS and CBL from organisms other than S. cerevisiae and E.coli, especially thermophiles, are expected to be sources of enzymeshaving even greater stability. The isolated enzymes can also be selectedfor a higher affinity to homocysteine. In addition, protein engineeringof the yeast CBS and bacterial CBL can lead to enzymes with improvedcommercial properties, including higher affinities, faster reactionrates, easier purification, and a longer half-life upon storage.

[0075] Similarly, cystathionine γ-lyase (CGL) has been described indetail in many species (see, for example, Yamagata, et al., J.Bacteriol., 175:4800-4808 (1993) incorporated herein by reference).Manipulation of the genes which encode CGL can be further modifiedgenetically by standard recombinant methods and inserted into a hostcell to develop higher level production strains.

[0076] Recombinant expression of the genes for CBS, CBL, and CGL aretypical. These methods can provide large quantities of purified enzymeand the genes can be modified, for example, to add a defined peptidesequence (an affinity tag) to the expressed enzymes that can be used foraffinity purification. Through the use of affinity tags in theexpression of the recombinant enzymes, the purification andimmobilization of these enzymes can be greatly simplified, leading tolower costs. Affinity tags are known in the art and include for example,but not limited, poly-histidine, GST (glutathione-S-transferase),strep-tag, “flag,” c-myc sequences, and the like. Tags are widely usedfor affinity purification of recombinantly expressed proteins, and manyare commercially available.

[0077] In a preferred embodiment, CBS and CBL are immobilized on a solidsurface in a manner that maintains the activities of the enzymes and ina manner that keeps homocysteine free to interact directly with theproteins. By immobilizing the enzymes at high densities, the diffusiondistances between the different proteins are reduced relative to enzymesin solution. As a result, the overall rate of reaction of the assayincreases greatly, since the product of one enzyme can be reacted uponby the other enzyme and vice versa. Acceleration of the reaction ratesis due to a local increase in the concentrations of the intermediates,which are in closer proximity to the next enzyme as a result ofimmobilization. Increasing the rate of the homocysteine assay can be animportant factor for the marketability of the diagnostics productbecause the K_(m) of CBS and of CBL are in the millimolar range but theblood concentrations of homocysteine are approximately 10 μM.Diffusion-limited reactions are known to require a high concentration ofthe enzymes to provide a rapid reaction rate. Immobilization of theenzymes in the cycling assay overcomes difficulties which might bepresented by the low affinities of these two enzymes for theirrespective substrates.

[0078] In yet another preferred embodiment CBS and CBL are immobilizedas a fusion protein. The distance and orientation of the two differentproteins relative to each other can be controlled during theconstruction of the fusion gene expression vector. By properlypositioning the two activities in a fusion protein, homocysteine andcystathionine interconversion can occur extremely rapidly virtuallywithin the protein, since diffusion distances can be minimized betweenthe two active centers. The homocysteine, therefore, behaves like acofactor in the overall enzymatic cycling reaction, which makes pyruvateand ammonia from L-serine.

[0079] The enzymatic cycling assay of the present invention can beprovided in a number of assay formats. The simplest format of the assayprovides a reagent solution comprising L-serine, CBS, or a derivativethereof, CBL, or a derivative thereof, and various buffers and auxiliarysubstances necessary for optimization of the enzyme reactions. In aparticularly preferred embodiment of the present invention CBS, or aderivative thereof, and CBL, or a derivative thereof, are providedimmobilized on a solid surface.

[0080] Immobilization of enzymes to a solid surface while retainingsubstantially all the activity of the enzyme are well known in the art.Several methods have been used for immobilization including, covalentbinding, physical absorption, entrapment, and electrochemicaldeposition. As one example glutaraldehyde has been used to immobilizecreatinine deaminase and glutamate oxidase to propylamine derivatizedcontrolled pore glass beads (Rui et al., Ann. NY Acad. Sci., 672:264-271(1992)). Other examples include, but are not limited to, the use ofcarbodiimides to covalently attach enzymes to succinate derivatizedglass beads (Rui et al., supra), and the use of hetero- orhomo-bifunctional cross-linking reagents (e.g., SPDP, N-succinimidyl3-(2-pyridyldithio)propionate; SMPT,succinimidyloxycarbonyl-α-methyl-α-(2-pyridyl-dithio)toluene; DSP,dithiobis(succinimidylpropionate); DTSSP,3,3′-dithiobis(sulfosuccinimidylpropionate); DSS, disuccinimidylsuberate, and the like) to covalently attach an enzyme through, forexample an amine or sulfhydryl group to a derivatized solid surface(Wilson, et al., Biosens. Bioelectro., 11:805-810 (1996)).

[0081] The “solid surface” as used herein can include a porous ornon-porous water insoluble material that can have any one of a number ofshapes, such as a strip, rod particle, including a bead or the like.Suitable materials are well known in the art and can include, forexample, paper, filter paper, nylon, glass, ceramic, silica, alumina,diatomaceous earth, cellulose, polymethacrylate, polypropylene,polystyrene, poly-ethylene, polyvinylchloride, and derivatives of eachthereof. The solid surface can be the sides and bottom of the testcontainer or can be a material added to the test container. In onepreferred embodiment the solid surface comprises a bead which is addedto the test container.

[0082] Both yeast and bacteria have enzymes that convert L-serine topyruvate, activities that are sometimes referred to as serine deaminaseor serine dehydratase. Well established mutagenic methods can be used togreatly reduce or eliminate these activities, which can contributedsignificantly to the background in the homocysteine cycling assay.Saccharomyces cerevisiae, in particular, may be genetically manipulatedto delete out the serine deaminase activities, which are necessary for(1) isoleucine synthesis and (2) growth on serine as a carbon ornitrogen source, by using a simple method known as “gene disruption.” Bydeleting the cha1 gene of yeast, a major serine deaminase activity isremoved, thereby eliminating one potential background problem to thecycling assay. In addition, the cha1 deletion can be useful forselecting CBS-CBL fusion proteins and for genetically engineering thosefusion proteins. The cha1 deletion strain is unable to grow on serine asthe sole carbon or nitrogen source. The CBS-CBL fusion gene is able tocomplement the deletion, so long as homocysteine is available as a“cofactor,” because the cycling system produces the same net result asthe serine deaminase. Therefore, the cha1 deletion can be used to selectfor and maintain improved CBS-CBL fusions, especially if a population ofthe fusion genes is mutated and placed on single-copy vectors or ontochromosomes in yeast. Cells that grow faster on serine media or have areduced requirement for homocysteine may contain mutations that code forfaster enzymes or proteins with higher affinities for homocysteine andcystathionine.

[0083] The present invention also provides, as an alternative method forassessing the amount of homocysteine in a sample, the use of ametabolite binding transcription factor. When a metabolite bindingtranscription factor is bound to their respective metabolite the factorcomplex binds to a specific receptor polynucleotide sequence. Thebinding of the transcription factor complex to its receptor consensussequence can be monitored and correlated to the amount of the metabolitepresent in the sample.

[0084] A number of transcription factors that bind to specific DNAbinding sites upon association with a small molecule or metabolite areknown in the art. For example, E. coli has an operon regulatory element,the transcription factor MetR which regulates the Met operon in responseto binding to homocysteine (Cai et al., Biochem. Biophys. Res. Commun.,163:79-83 (1989)). MetR or derivatives thereof regulate genes such asMetE (cobolamin independent methionine synthase), MetH (cobolamindependent methionine synthase) and GlyA (serine hydrogenase). MetR andits derivatives preferentially bind to its consensus DNA sequence in thepresence of homocysteine and is free in the cell cytoplasm in itsabsence. The binding of MetR in the presence of homocysteine can beexploited to assess the amount of homocysteine in a sample suspected ofcontaining homocysteine.

[0085] In a particular embodiment of the invention the assay comprises apolynucleotide sequence encoding the consensus sequence for a receptorpolynucleotide for MetR immobilized on a solid support in a manner whichallows for the accessibility of the consensus receptor sequence forbinding to MetR. The nucleotide sequence can be, for example:GTTAATGTTGAACAAATCTCATGTTGCGTG (SEQ ID NO: 11)

[0086] A sample, such as plasma, blood or urine, is treated to releaseany protein bound homocysteine prior to admixing the sample with areagent solution comprising MetR in a buffering agent in the presence ofthe immobilized consensus receptor sequence under conditions conduciveto complex formation. After an incubation period the solid support iswashed to remove unbound reagents and the amount of MetR that remainsbound to the solid support is assessed. The determination of the amountof MetR that remains bound to the solid surface typically can be anELISA, surface plasmon resonance, i.e., BIACORE, or simply a standardprotein assay, and this amount is correlated with the amount ofhomocysteine present in the sample.

[0087] Treatment of the sample to release homocysteine can beaccomplished with a reducing agent. As described above a number ofagents are well known in the art. Typically a sample will be admixedwith a buffer solution containing a sufficient amount of reducing agentto release substantially all of the homocysteine from any associatedproteins present in the sample. Commonly used reducing agents include,but are not limited to, sodium and potassium borohydride,β-mercaptoethanol, dithiothreitol, dithioerythritol, thioglycolic acidor glutathione; or a phosphine or a trialkylphosphine, such astributylphosphine and tris(2-carboxyethyl)phosphine (TCEP), and thelike.

[0088] The MetR protein can also be modified to incorporate a detectablelabel, such as a chromophore, a fluorophore, and the like, to improvethe speed and sensitivity of the assay. The addition of the detectablelabel can reduce the time period required to run the assay byeliminating certain sample handling steps, for example, additionalwashing steps. Incorporation of a fluorophore that emits light at thewavelength absorbed by oligonucleotides into the consensus bindingsequence allows for the measurement of fluorescent energy transfer toquantify the binding of the protein to the consensus sequence. Methodswhich can be used to measure fluoresence transfer include, for example,Fluorescence resonance energy transfer (FRET) or a proximityscintillation assay. Additional modification of the MetR bindingconsensus sequence by the addition of an additional detectable labelwithin the binding domain can also improve the specificity of the assay.

[0089] Additionally, a defined peptide sequence (an affinity tag) can beadded to the MetR protein to aid in purification and detection. Affinitytags are known in the art and include for example, but not limited,poly-histidine, strep-tag, “flag,” c-myc sequences, and the like. Tagsare widely used for affinity purification of recombinantly expressedproteins, and many are commercially available. Further, mutationalalterations to improve binding affinity of a recombinantly expressedMetR can be used to provide additional assay reagents.

[0090] In a separate embodiment of the present invention, the amount ofhomocysteine in a sample can be determined by the enzymatic utilizationof homocysteine in the sample. Conversion of homocysteine in cellularmetabolism typically releases a proton from the sulfhydryl residue ofhomocysteine. Release of this proton was used to measure the bindingaffinity of homocysteine to E. coli cobolamin dependent methioninesynthase (Jerret et al., Biochemistry, 36:15739-15748 (1997)). Enzymereactions catalyzed by proteins, such as methionine synthase, generallyrequire cofactors or additional substrates to complete the reaction. Ifthese cofactors and substrates are not present, the homocysteine remainsbound to the enzyme. In the present method, measurement of the releasedproton removes the proton released by the enzymatic reaction driving thereaction toward consumption of the homocysteine present in the sample.The measurement of the change in pH associated with the released protonsprovides a determination of the amount of homocysteine in the sample. Ina preferred embodiment, for example, cobolamin independent methioninesynthase, can be used in the assay.

[0091] As a matter of convenience, the reagents for use in the presentinvention can be provided in a test kit for use in the assay method. Atypical kit comprises a packaged combination of a container to hold thetest solution, L-serine, CBS, or a derivative thereof, and CBL, or aderivative thereof, and auxiliary buffers and substances required foroptimal reaction conditions. Importantly, a variety of preservativesand/or stabilizing agents may also be included with any/all kit reagentsand/or enzymes to elongate shelf life as well as “on-line” life forsample testing. The kit can further comprise reducing agents or CGL forpretreating the sample prior to the cycling assay. Further, the kit caninclude lactate dehydrogenase, NADH and a dye capable of an observablecolor change upon oxidation.

[0092] Under appropriate circumstances one or more of the reagents inthe kit can be provided in solution (with or without preservativesand/or stabilizing agents) or as a dry powder, usually lyophilized,including excipients, preservatives, and/or stabilizing agents, which ondissolution can provide for a reagent solution having the appropriateconcentrations for performing the assay in accordance with the presentinvention. To enhance the versatility of the subject invention, thereagents can be provided in packaged combination, in the same orseparate containers, so that the ratio of the reagents provides forsubstantial optimization of the method and assay. The reagents can eachbe in separate containers or various reagents can be combined in one ormore containers depending on the stability of the reagents. As a matterof convenience, the reagents employed in the assay method of the presentinvention can be provided in predetermined amounts. The test kit canalso contain written instructions on how to use the reagents and/or howto perform a particular assay, for example in the form of a packageinsert.

[0093] The following examples are offered by way of illustration, not byway of limitation.

EXAMPLE 1 Cloning and Expression of the E. coli Cystathionine β-LyaseGene

[0094] In this example standard PCR procedures were used to clone the E.coli CBL gene. The isolated gene was inserted into an expression vectorwith an affinity tag sequence (6HIS), and the expressed protein waspurified with an affinity column. A substantial amount of the CBL enzymeactivity in the host cells transformed with the expression vectorconstruct was purified from the cell lysates.

[0095] Cloning of the E. coli Cystathionine β-lyase (CBL, EC 4.4.1.8)Gene

[0096] Standard PCR procedures using EXPAND HIGH FIDELITY PCR system(Roche Biochemicals) were employed to amplify the E. coli CBL gene usingONE SHOT TOP 10 competent E. coli cells (Invitrogen) as the genomic DNAsource. PCR primer sequences for CBL cloning were as follows: (CBLN-terminal primer: 5′-CGACGGATCC GATGGCGGACAAAAAGCTTG-3′; SEQ ID NO: 1)and (CBL C-terminal primer: 5′-CGCAGCAGCT GTTATACAATTCGCGCAAA-3′; SEQ IDNO: 2). The PCR amplified CBL gene product was then purified by agarosegel electrophoresis, digested with BamHI and PvuII restrictionendonucleases, and purified again by agarose gel electrophoresis. Thepurified E. coli CBL gene was then ligated into gel purified,BamHI/PvuII digested pRSETB protein expression vector (Invitrogen). Theresulting construct provided the CBL gene under control of the T7transcriptional promoter, cloned in frame with an amino terminal sixHistidine (6HIS) residue leader sequence (hereafter referred to as6HISCBL) which allows for convenient expression, affinity purification,and immunostaining analysis of recombinant 6HISCBL protein from E. colicells. The DNA sequence of E. coli Cystathionine β-lyase (CBL) gene ispresented (SEQ ID: 21). The protein sequence of the cloned E. coliCystathionine β-lyase (CBL) having an amino-(NH3)-terminal 6Histidine(6His) affinity tag, resulting from cloning the CBL gene into the pRSETBbacterial expression plasmid is also presented (SEQ ID: 19).

[0097] Cystathionine β-Lyase Protein Expression and Purification

[0098] The pRSETB(6HISCBL) plasmid construct was transformed into BL21(DE3)pLysS E. coli strain (Invitrogen), spread on LB/Agar platescontaining (100 μg/ml ampicillin, 35 μg/ml chloramphenicol), and wasthen incubated overnight at 37° C. Single bacterial colonies were thentransferred and grown overnight in flasks containing 100 ml LB growthmedium containing ampicillin and chloramphenicol. The 100 ml overnightculture was then added to multiple flasks containing 1 L of fresh LBgrowth medium containing ampicillin, and allowed to grow until reachingan A₆₀₀ of 0.6 (approx. 2.5 hours). In order to induce CBL proteinexpression (driven by the T7 promoter),isopropyl-β-D-thiogalactopyranoside (IPTG) was then added to the growthmedium to a 0.1 to 1 mM final concentration. The CBL enzyme cofactorpyridoxal 5′-phosphate (PLP) was also added to a final concentration of100 μM in the growth medium during induction. After monitoring cellgrowth and 6HISCBL protein expression over a 5 hour period, E. colicells were harvested by centrifugation, resuspended, and lysed by theaddition of BUGBUSTER Protein Extraction Reagent (Novagen Inc.) in thepresence of EDTA free complete protease inhibitor (Roche Biochemicals)as described in the manufacture's protocol. The cell lysate was thentreated with DNase I, cleared by centrifugation and loaded onto anaffinity column containing Poly-His Protein Purification Resin (RocheBiochemicals). The column was then washed with 10 bed volumes ofequilibrating buffer (50 mM KPi, pH 7.8, 0.5 M NaCl), followed byelution with 5 bed volumes of elution buffer 1 (50 mM KPi, pH 7.8, 0.5 MNaCl, 10 mM imidazole; EB 1), 5 bed volumes of elution buffer 2 (50 mMKPi, pH 7.8, 0.5 M NaCl, 50 mM imidazole; EB2), and 5 bed volumes ofelution buffer 3 (50 mM KPi, pH 7.8, 0.5 M NaCl, 500 mM imidazole; EB3).Samples taken during each step of the purification procedure were thenanalyzed by denaturing protein gel electrophoresis (SDS-PAGE) andstained with either Coomassie Brilliant Blue, or subjected to Westernblot analysis using a monoclonal anti-polyhistidine peroxidaseconjugated mouse immunoglobulin for detection as described by themanufacturer (Sigma).

[0099] The purified 6HISCBL protein was found to elute primarily inelution buffers 2 and 3 (EB2 and EB3). The CBL containing eluate wasthen concentrated using centrifugal filtration chambers (Amicon) anddialyzed overnight at 4° C. against storage buffer (50 mM Tris HCl, pH8, 100 mM NaCl, 5 mM PLP), aliquoted and stored frozen at −80° C. Theconcentration of purified homogenous CBL enzyme was then calculated onthe basis of the extinction coefficient at 280 nm and a subunit weightof 43,312 Da. (Clausen et al. Biochem. 36:12633-42 (1997)). A typicalyield of purified 6HISCBL was in the range of 20-30 mg 6HISCBL per gramof E. coli cell paste.

[0100] CBL Activity Assay

[0101] The CBL activity of affinity purified recombinant 6HisCBL proteinexpressed from the pRSETB(6HisCBL) plasmid construct was performedessentially as described previously (Clausen et al., Biochem.36:12633-42 (1997) incorporated herein by reference). Briefly, assaymixtures contained 100 mM Tris/HCl (pH 8.5), 5 mM L-cystathionine, 1 mM5,5′-dithiobis(2-nitrobenzoic acid) (DTNB; Ellman's reagent), and6HisCBL enzyme in a final reaction volume of 1 mL. The reaction wasfollowed by recording the increase in absorbance at 412 nm with time.The same enzyme reaction was also carried out on samples having knownconcentrations of substrate to produce a standard curve. Substantiallyall of the CBL activity detected in the whole cell lysate was recoveredand found present in the experimentally purified samples of recombinant6HisCBL enzyme produced from the pRSETB(6HisCBL) plasmid construct.

EXAMPLE 2 Cloning and Expression of the Yeast Cystathionine β-Synthase(CBS) Gene in Yeast

[0102] In this example PCR methods were used to clone the CBS gene fromyeast. The PCR fragment comprising the CBS gene was inserted into anexpression vector both with and without an affinity tag which was addedto assist in purification of the expressed gene product.

[0103] A laboratory strain of Saccharomyces cerevisiae (5153-11-1 (his3met2)) was used as the source of the CBS gene. The cells were grown onYEPD at 32° C., spun down, washed with water, and transferred into 200μl of Yeast Plasmid Buffer (100 mM NaCl, 10 mM Tris (pH 8), 1 mM EDTA,0.2% SDS). The cell suspension was mixed with an equal volume of 0.45 mmglass beads and vortexed at high speed for 3×30 seconds. The liquid wasremoved by pipetting and centrifuged for two minutes in a microfuge toremove cell debris. The supernatant was extracted with PCI(phenol:chloroform:isoamyl alcohols (25:24:1) and chloroform. Sodiumchloride (5 M) was added (about 20 μl), and the DNA was precipitatedwith an equal volume of isopropanol.

[0104] This genomic yeast DNA was amplified by PCR with primers CI-001(CGGGGATCCTGATGCATGCATGATAAGGA; SEQ ID NO: 3) and CI-012 (CGGCATTACCGTTTCACTAATTTATTG; SEQ ID NO: 4), which are published nucleotide sequencesthat flank the structural gene for the yeast CBS. Primer CI-001 (SEQ ID:3) contains a BamHI site found on the untranslated 3′ side of the CBSgene. To add a BamHI sequence to the 5′ side of the PCR fragment, thegene was further amplified by PCR with CI-001 (SEQ ID NO: 3) and CI-002(GTTGGATCCGGCATTACCGTTTCAC; SEQ ID NO: 5). The resulting PCR product wasa DNA sequence of approximately 1937 base pairs, including 1521 basepairs that encode the CBS protein. The additional DNA that flanks thecoding sequence comprises the natural promoter and transcriptiontermination sequences of the yeast CBS gene.

[0105] A stronger promoter was inserted upstream of the CBS gene topromote higher levels of protein expression. The yeast TPI1 promoter wascloned from the same genomic yeast DNA using the primers CI-009(GGTGGATCCATCAATAGATACGTA CGTCCT; SEQ ID NO: 6) and CI-010(TTTTAGTTTATGTATGTGTTTTTTG; SEQ ID NO: 7) in a standard PCR reaction. Toattach the CBS coding region to the TPI1 promoter, an adapter primer isused: CI-011 (AACACATACATAAACTAAAAATGACT AAATCTGAGCAGCAA; SEQ ID NO: 8),which contains 20 bases of the TPI1 promoter and the first 21 bases ofthe CBS gene. The CBS sequence obtained above was amplified by PCR withprimers CI-001 (SEQ ID NO: 3) and CI-011 (SEQ ID NO: 8). The resultingPCR product was mixed with the TPI1 promoter product from above, and themixture was amplified by PCR with primers CI-001 (SEQ ID NO: 3) andCI-009 (SEQ ID NO: 6) to produce a fusion gene designated “TPICBS,” thatcomprising the TPI1 promoter, the CBS coding sequence, and the CBStranscription terminator.

[0106] The CBS and TPICBS PCR products each comprise BamHI sites on boththe 5′ and 3′ sides of the coding region. These PCR products werepurified with a PCR WIZARD PREP (Promega), according to themanufacturer's recommendations, and were cut with BamHI. The resultingBamHI nucleotide fragments were run on a 1% Agarose TBE Gel and purifiedwith NA45 paper (S&S). The yeast, E. coli shuttle vector C1-1 has aunique BamHI site in the gene for tetracycline resistance was used forexpression of the CBS gene. C1-1 vector was cut with BamHI and treatedwith shrimp alkaline phosphatase, according to the manufacturer'sinstructions (Roche Biochemicals). The CBS and TPICBS BamHI fragmentswere ligated into the linearized C1-1 vector. The ligation reaction wastransformed into TOP 10 cells (Invitrogen), which were plated onto LBampicillin plates. Amp-resistant transformants were transferred onto LBamp and LB tet plates to determine tetracycline sensitive clones.Several tet-sensitive colonies were selected from the LB amp plates,grown overnight on LB amp plates, and were further processed by thealkaline lysis method for plasmid DNA. The DNA was cut with BamHI andseparated on a 1% agarose gel to determine which plasmids contain theCBS or TPICBS inserts.

[0107] C1-1 plasmids that carry inserts were transformed into the yeaststrain, INVSc-1, a diploid strain that is commercially available fromInvitrogen. INVSc-1 has a mutation in both copies of the LEU2 gene andrequires the LEU2 gene from the C1-1 plasmid for selective growth onleucineless media. INVSc-1 was transformed with the C1-1 relatedplasmids, using the Yeast Transformation Kit from Invitrogen, accordingto the manufacturer's instructions. This kit involved makingspheroplasts of the cells with Zymolyase, followed by a calciumtreatment for DNA transformation. The transformants were plated onto asynthetic medium containing 1 M sorbitol, yeast nitrogen base, and yeastsupplements, excluding leucine. Transformants were visible in theoverlay agar after three days of incubation at 32° C.

[0108] Attachment of Poly-Histidine to Cystathionine α-Synthase forYeast Expression

[0109] DNA sequences that encode six histidines were added to the 3′ endof the CBS coding region, in both the CBS and TPICBS vector constructs.Poly-histidine coding sequences were added to the original CBS clone byamplification of the CBS PCR fragment from above with the primer CI-017(ATGATGATGATGATGATGACCTGCT AAGTAGCTCAGTAA; SEQ ID NO: 9) and primerCI-002 (SEQ ID NO: 5). The resulting PCR product comprises the naturalCBS promoter region, the CBS coding region, and a further codingsequence for glycine and six histidine residues. The glycine residue wasadded to increase the spatial flexibility of the poly-his peptide aftertranslation. The poly-his CBS PCR product was gel-purified away from theoriginal CBS DNA and was amplified by PCR sequentially twice thereafterwith primers, CI-002 (SEQ ID NO: 5) and CI-017 (SEQ ID NO: 9) tovirtually eliminate the original CBS DNA.

[0110] Similarly, the TPICBS PCR product from above was amplified withprimers, CI-009 (SEQ ID NO: 6) and CI-017 (SEQ ID NO: 9). The PCRproduct was gel purified and reamplified twice with CI-009 (SEQ ID NO:6) and CI-017 (SEQ ID NO: 9) to greatly reduce the input TPICBS DNAsequence.

[0111] The CBS gene was also cloned by PCR with primers CI-001 (SEQ IDNO: 3) and CI-018 (CATCATCATCATCATCATTAAATAAGAACCCACGCT; SEQ ID NO: 10)to produce a poly-histidine sequence with a TAA stop codon, followed bythe transcription terminator sequence. This short PCR product(“terminator”) of about 190 bp was also gel purified and cloned by PCRwith the same primers. The poly-his CBS and “terminator” fragments werecombined into one PCR reaction with the primers, CI-001 (SEQ ID NO: 3)and CI-002 (SEQ ID NO: 5). The resulting amplification produced anucleic acid molecule designated “CBSH,” comprising the natural CBSpromoter, the entire CBS coding region, one additional glycyl residue,six histadyl residues with a stop codon, and the CBS transcriptionterminator.

[0112] Likewise, the poly-his TPICBS and terminator fragments werecombined into one PCR reaction with the primers, CI-001 (SEQ ID NO: 3)and CI-009 (SEQ ID NO: 6). The resulting nucleic acid product,designated “TPICBSH” was the same as the CBSH sequence, except that theTPI1 promoter replaced the natural CBS promoter. The CBSH and TPICBSH(each containing a poly-histidine affinity tag sequence) were cut withBamHI and ligated into C1-1, as done above for the CBS and TPICBSsequences. The ligation mixes were transformed into INVSc-1.Tet-sensitive colonies were examined for the insertion of theappropriate cloned genes into C1-1 by restriction enzyme analysis.

EXAMPLE 3 Cloning, Expression, and Purification of the YeastCystathionine β-Synthase (CBS) Gene in E. coli

[0113] In this example standard PCR and molecular biology techniqueswere used to clone the Saccharomyces cerevisiae (INVSc1) Cystathionineβ-Synthase (CBS) gene as a fusion protein with an amino-terminalglutathione S-transferase (GST). Addition of this region of glutathioneS-transferase allows for the single-step affinity purification of theCBS protein from the E. coli culture medium and may also assist insolubilization and/or to promote proper refolding of the enzyme in anactive form.

[0114] Cloning

[0115] The laboratory strain of Saccharomyces cerevisiae (INVSc 1)(Invitrogen) was used as the source of CBS gene. A single colony ofINVSc1 yeast was boiled in 100 μl of deionized water for 5 min. andvortexed rapidly in the presence of an equal volume of 0.45 mm glassbeads for 2 min. One microliter of this cell lysate was then used as thesource of DNA for PCR amplification of yeast CBS gene using primersCI-024 (GCGGGTCGACTATGACTAAATCTGAGCAGCAAGCC; SEQ ID NO: 12) and CI-025(GCGTGCGGCCGCGTTATGCTAAGTAGCTCAG; SEQ ID NO: 13) which contain flankingsequences to the published yeast CBS gene and restriction sites forcloning into the pGEX-6P-2 (Amersham Pharmacia) expression vector. Theresulting PCR product (approximately 1548 bp) was then isolated andpurified by agarose gel DNA extraction using a commercial kit (Roche).The purified PCR product containing the CBS gene flanked by SalI (5′end)and NotI (3′ end) restriction enzyme sites was then digested (by SalIand NotI), gel purified, and ligated into the pGEX6P-2 expression vector(Amersham Pharmacia). Restriction enzyme mapping was used to confirm theidentity of the PCR amplified DNA as the yeast CBS gene. The DNAsequence for the cloned Saccharomyces cerevisiae Cystathionineβ-Synthase (CBS) gene is depicted (SEQ ID: 14). The protein sequence ofthe cloned Saccharomyces cerevisiae CBS having an amino-(NH3)-terminalGST fusion protein attached as a result of cloning into the bacterialexpression vector pGEX6P-2 is also presented (SEQ ID: 20).

[0116] Expression and Purification

[0117] The pGEX6P-2 expression vector containing the cloned yeast CBSgene (described above) was transformed into TOP 10 E. coli cells(Invitrogen). Single colonies were picked and cell cultures were grownin LB media containing 100 μg/ml ampicillin at 30EC until reaching anabsorbance at 600 nm of approx. 0.5 OD. In order to induce expression ofthe GST-CBS fusion protein, (driven by the tac promoter),isopropyl-β-D-thiogalactopyranoside (IPTG) was added to the growthmedium to a final concentration of 0.1 mM. The CBS cofactor, pyridoxal5′ phosphate, was also added to the growth medium at 100 μM finalconcentration during induction. Induced cells were then allowed to growat 30° C. for an additional 21 hrs before harvesting by centrifugation.

[0118] Affinity purification of the GST-CBS protein using GlutathioneSepharose 4B affinity resin was then performed essentially as describedin the manufacture's protocol (Amersham Pharmacia). Briefly, cells weresuspended in phosphate buffered saline (PBS) and disrupted by sonicationon ice. TritonX-100 was then added to a final concentration of 0.1%(v/v), and the sonicate was shaken at room temperature of 30-45 min. Thecell lysate was then cleared of insoluble debris by centrifugation,treated with DNase I, and loaded onto a column of Glutathione Sepharose4B affinity resin. Nonspecifically bound proteins were washed away with10 column volumes of PBS, and the desired GST-CBS protein was theneluted from the affinity resin by the addition of 5 column volumes ofelution buffer (50 mM Tris, pH 8, 1.4 mM P-mercaptoethanol (BME), 100 mMNaCl, 0.1% (v/v) TritonX-100, 50 mM reduced glutathione). Purificationof the GST-CBS fusion protein was then confirmed by Coomassie Bluestained SDS-PAGE (sodium dodecylsulfate, polyacrylamide gelelectrophoresis). The eluted GST-CBS was then dialyzed into storagebuffer (50 mM Tris HCl, pH8, 100 mM NaCl, 5 μM PLP), aliquoted andstored at −80° C. The concentration of purified GST-CBS was thendetermined using the MICRO BCA Protein Assay Kit (Pierce Chemical). Atypical yield of purified GST-CBS was in the range of 5-10 mg GST-CBSper gram of E. coli paste.

[0119] Activity Assay for CBS

[0120] The CBS activity of the purified yeast GST-CBS fusion protein wasperformed as described by Kashiwamata and Greenberg (Biochim. Biophy.Acta 212:488-500 (1970)). This assay is based on the ability of CBS tosynthesize cystathionine from serine and homocysteine. The amount ofcystathionine formed by CBS was then determined spectrophotometricallyby detecting the ninhydrin reaction chromagen at 455 nm and compared toa standard curve generated using samples of known cystathionineconcentrations. CBS activity was also determined by the enzymatic assaysdescribed infra, or by other assays well known to the skilled artisan.

EXAMPLE 4 Construction of ADH2CYS4 Fusion Construct Expression andPurification of Yeast Cystathionine β-Synthase in Yeast

[0121] In this example a strong promoter, ADH2, was inserted upstream ofthe CYS4 gene and constructed as a fusion with the yeast CBS gene topromote higher protein expression. The ADH2 promoter is regulated by theAdr1p, and is repressed when yeast cells are grown in glucose containingmedium and becomes derepressed when the cells are grown in anon-fermentable carbon source. This allows for regulated expression, ifoverproduction of a protein is lethal to the cell.

[0122] Construction of ADH2CYS4 Gene

[0123] Yeast genomic DNA was amplified with primers C₁₀₂₉(CTATATCGTAATAATGACTAAATCTGAGCAGCAAGCCGATTCA; SEQ ID NO: 15) and CI-017(ATGATGATGATGATGATGACCTCGTAAGTAGCTCAGTAA; SEQ ID NO: 9) to produce allof the CYS4 gene without the stop codon and transcription terminator.CI-029 (SEQ ID NO: 15) primer contains the last 13 bases of the ADH2promoter region cloned in addition to the first ten codons of CYS4 gene.Primer CI-017 (SEQ ID NO: 9) has the sequence for the last six codons ofCYS4 gene, except the stop codon plus a glycyl residue and six histadylresidues. The resulting PCR product comprises the CYS4 coding region,and a further coding sequence for glycine and six histidine residues.The glycine was added to increase the spatial flexibility of thepoly-his peptide after translation. The “terminator” fragment wasproduced by amplifying the same genomic DNA with primers CI-018(CATCATCATCATCATCATTAAATAAG AACCCACGCT; SEQ ID NO: 10) and CI-040(AGGGCTCGAGGATCCCGGGGGT GCTATTATGAATGCACGG; SEQ ID NO: 16) to produce apoly-histidine sequence with a TAA stop codon, followed by thetranscription terminator. The CI-029/CI-017 PCR reaction produced anapproximately 1530 bp fragment while CI-018/CI-040 reaction produced anapproximately 331 bp fragment. These two fragments were joined by mixingtheir PCR products and amplifying with primers CI-029/CI-040. Theresulting product was designated CYS4H and was approximately 1861 bp inlength.

[0124] To join the ADH2 promoter to the CYS4H gene fragment, a PCRproduct was first generated with primer CI-027(CGGGGATCCGACGTTTTGCCCGCAGGCG GGAAACC; SEQ ID NO: 17) and primer CI-028(AGATTTAGTCATTATTACGATAT AGTTAATAGTTGATAG; SEQ ID NO: 18) from the sameyeast genomic DNA to produce the ADH2 promoter sequence. Primer CI-028(SEQ ID NO: 18) contains the first 12 nucleotides of the coding regionof CYS4 gene. Therefore, between primers CI-028 (SEQ ID NO: 18) andCI-029 (SEQ ID NO: 15) there are 25 nucleotides of complimentarity DNAin their respective products. The ADH2 PCR product was mixed with CYS4Hfragment and amplified with primers CI-027 (SEQ ID NO: 17) and CI-040(SEQ ID NO: 16), to produce a product fragment of approximately 2201 bpdesignated ADH2CYS4H. Each of these PCR products was confirmed byrestriction endonuclease analysis.

[0125] The ADH2CYS4H product was cloned into YEp24 by cutting both DNAwith BamHI and ligating using T4 DNA ligase. Ligated products weretransformed into E. coli (TOP 10; Invitrogen) cells and plated onLB-agar plus ampicillin plates. Plasmids, prepared using PlasmidPreparation kit (Roche) according to the manufacture's recommendationfrom putative recombinants, were analyzed by restriction endonuclease.The plasmids from clones found to have the correct insert weretransformed into S. cerevisiae (BJ5460) and plated on synthetic completemedium minus uracil (SC-ura), supplemented with 2% glucose. After twodays putative recombinants were re-streaked on the same type of platesto isolate colonies. The cells are induced by growing in SC-ura plus 2%ethanol and 1% peptone, usually supplemented with 0.1% glucose to enablethe cells to grow within 24 hours. Following overnight growth cells wereare collected by centrifugation and the cell pellet was saved at −70° C.until lysis. Cells were lysed by resuspending the cell pellet in 0.05volume of the original volume in lysis buffer. Glass beads were thenadded to the meniscus of the buffer.

[0126] The whole suspension was vortex for 1 min each for four times.Cells were cooled on ice for 1 min between vortexing. Lysate wastransferred to fresh tube and centrifuged for 10 min at 10,000×g. Thecleared lysate was layered onto a His-Resin column (Roche) and theprotein was purified according to manufacture's recommendation. Apartially purified protein was recovered in elution three.

EXAMPLE 5 Examples of Non-Cycling Portion of Assay Formats forHomocysteine

[0127] In this example the non-cycling portion of the assay for pyruvateand cystathionine are demonstrated.

[0128] Pyruvate Assay:

[0129] Pyruvate: The present invention provides for the measurement ofhomocysteine based on the use of the enzymes CBS and CBL to cyclehomocysteine with the production of pyruvate and ammonia. The rate ofpyruvate and ammonia production was predicted to be proportional to theoriginal homocysteine concentration in the sample. Measurement of thisrate using appropriate enzyme(s) presents an opportunity for ahomogeneous homocysteine method.

[0130] In one aspect of the present invention a highly sensitive methodfor pyruvate quantitation has been developed. In the method pyruvateoxidase converts pyruvate to acetyl phosphate, carbon dioxide andperoxide. Peroxidase subsequently converts the peroxide, TOOS, and4-aminoantipyrine to a chromogen, which exhibits an absorption maximumnear 550 nm. The molar absorptivity of this chromogen is quite high,about 36 L mol-1 cm-1.

[0131] Using reagent components shown in Table 1 and the Cobas FARA(Roche, Basel, Switzerland) instrument parameters listed in Table 2,pyruvate was easily measured in the μM range by an early-read blank andendpoint reaction in about 5-10 minutes. Absorbance versus time plotsfor aqueous calibrators are shown in FIG. 1. An actual calibration curveis shown in FIG. 2. TABLE 1 Pyruvate Method: Reagent Components ChemicalConcentration Reagent 1 HEPES, hemisodium salt 21.5 mM HEPES, Acid 28.6mM EDTA (4Na) 5.0 mM Mg SO₄.7H2O 49.8 mM K₂HPO₄, dibasic 7.5 mM TOOS 1.4mM TTHA 0.8 mM TPP 0.2 mM 4-aminoantipyrine 1.0 mM BSA 1.9 g/L Potassiumferrocyanide 0.07 mM Peroxidase, horseradish 3.0 KU/L Pyruvate Oxidase3.0 KU/L pH = 7.0

[0132] TABLE 2 Pyruvate Method: Cobas FARA parameters Parameter SettingGENERAL measurement mode ABS reaction mode P-A Calibration mode SLOPEAVG reagent blank REAG/DIL Wavelength 550 nm Temperature 37° C. ANALYSISsample volume 40 μL diluent volume 10 μL diluent name H₂O Reagent volume250 μL  incubation time 60 s start reagent volume na diluent name H₂Odiluent volume na incubation time na start reagent 2 volume na diluentname H₂O diluent volume na Temperature delay na ABSORBANCE READINGSFirst 0.5 sec Number 14  Interval  30 sec CALCULATION Number of steps 1calculation step A KINETIC First 1 Last 14  CALIBRATION Number of stds 1STD1 50 μM Replicate 2

[0133] Cystathionine Assay:

[0134] Cystathionine was measured by adding CBL to the pyruvate reagentdescribed above. CBL converts cystathionine to homocysteine, pyruvateand ammonia. The pyruvate was then measured as described above. Thecomposition of the reagent used to measure cystathionine is shown inTable 3. The measurement can be made using any suitable instrument. Inthe present example a Cobas FARA (Roche, Basel, Switzerland) was used.Typical reaction parameters are shown in Table 4. FIG. 3 depictsabsorbance vs. time plots obtained using aqueous solutions ofcystathionine. The reaction reaches endpoint in less than ten minutes.TABLE 3 Cystathionine Method: Reagent Components Chemical ConcentrationReagent 1 HEPES, hemisodium salt 20.3 mM HEPES, Acid 27.0 mM EDTA (4Na)4.7 mM Mg SO4.7H2O 47.1 mM K2HPO4, dibasic 7.1 mM TOOS 1.3 mM TTHA 0.8mM TPP 0.2 mM 4-aminoantipyrine 0.9 mM BSA 1.8 g/L Potassiumferrocyanide 0.07 mM Peroxidase, horseradish 2.8 KU/L Pyruvate Oxidase2.8 KU/L CBL 1.9 g/L pH = 7.0

[0135] TABLE 4 Cystathione Method: Cobas FARA parameters ParameterSetting GENERAL measurement mode ABS reaction mode P-A calibration modeLIN INTER reagent blank REAG/DIL wavelength 550 nm temperature 37° C.ANALYSIS sample volume 30 μl diluent volume 10 μl diluent name H₂Oreagent volume 260 μl  incubation time 60 sec start reagent volume nadiluent name H₂O diluent volume na incubation time na start reagent 2volume na diluent name H₂O diluent volume na temperature delay naABSORBANCE READINGS first  5 sec number 21  interval 60 sec CALCULATIONnumber of steps 1 calculation step A ENDPOINT first 1 last 10 CALIBRATION number of stds 1 STDI 50 μM replicate 2

[0136]FIG. 4 depicts standard curves obtained using aqueous solutions ofcystathionine. These curves are linear over at least the cystathionineconcentration range of 0-100 μM. The chromophore formed was stable overapproximately 15-20 minutes. However, if samples containing 10-20 foldhigher cystathionine concentrations were assayed, some instability ofthe chromophore was evident.

[0137] In some experiments serine was included in the reaction mixture.Serine at a level of 250 μM exhibited no significant effect. Higherlevels of serine were also tolerated by this assay. The non-interferenceof serine is an important observation because the presence of serine isnecessary for the assay of homocysteine.

EXAMPLE 6 Enzyme Cycling Assays for Homocysteine

[0138] In this example the enzyme cycling systems for determining theconcentration of homocysteine are provided using either pyruvateoxidase/peroxidase signaling system or lactate dehydrogenase signalingsystem.

[0139] Demonstration of Cycling: CBS/CBL/PO/HRP Enzyme Cycling System

[0140] The cycling system has been demonstrated wherein the reaction wasinitiated using either homocysteine or cystathionine. The reagentcomponents used in these experiments are shown in Table 5. CBS andserine were added separately following mixing of the sample with reagent1 which contains all other components. Pertinent Cobas FARA parametersare shown in Table 6. Cystathionine or homocysteine was dissolved inwater to prepare standards at appropriate concentrations. Time versusabsorbance plots are shown in FIGS. 5 and 6. After a lag time of threeto six minutes the plots are linear. A typical homocysteine calibrationcurve (FIG. 7) which, while not completely linear, can be used toquantify homocysteine concentrations in suitable specimens using anappropriate curve-fitting technique. TABLE 5 CBS/CBL Cycling, PO/HPRMethod Reagent Components Chemical Concentration Reagent 1 HEPES,hemisodium salt 20.3 mM HEPES, Acid 27.0 mM EDTA (4Na) 4.7 mM MgSO₄.7H2O 47.1 mM K₂HPO₄, dibasic 7.1 mM TOOS 1.3 mM TTHA 0.8 mM TPP 0.2mM 4-aminoantipyrine 0.9 mM BSA 1.8 g/L Potassium ferrocyanide 0.07 mMPeroxidase, horseradish 3.0 KU/L Pyruvate Oxidase 3.0 KU/L CBL pH = 7.0Reagent SR1 Serine 3.1 mM Reagent SR2 CBS 1.23 g/L

[0141] TABLE 6 CBS/CBL/PO/HPR cycling system: Cobas FARA parametersParameter Setting GENERAL measurement mode ABS reaction modeP-I-SR1-I-SR2-A Calibration mode LIN INTER reagent blank REAG/DILWavelength 550 nm Temperature 37° C. ANALYSIS sample volume 30 μldiluent volume 10 μl diluent name H₂O reagent volume 260 μl  Incubationtime  60 sec start reagent volume 30 μl (serine) diluent name H₂Odiluent volume  5 μl Incubation time  10 sec start reagent 2 volume 30μl (CBS) diluent name H₂O diluent volume  5 μl temperature delay naABSORBANCE READINGS First 0.5 sec Number 14  Interval  30 secCALCULATION number of steps 1 Calculation step A ENDPOINT or KINETICFirst 1 Last 14  CALIBRATION number of stds 1 STD1 50 μM Replicate 2

[0142] Reduction of Disulfide Bonds:

[0143] Very little homocysteine is present in the reduced, nondisulfidebonded state in human plasma. Most homocysteine is coupled to proteinsor small molecules through disulfide bonds. Disulfide reduction of thesecompounds is necessary to liberate homocysteine for measurement by anymethod for total homocysteine determination. This example demonstratesthat use of a reducing agent is compatible with a system using lactatedehyrdrogenase and NADH. Potential candidate reducing agents includeDTE, DTT, n-acetylcysteine, thioglycolic acid, TCEP and the like. Foruse of these compounds in the method of the present invention, preciseadjustment of reducing agent concentration and instrument parameters arenecessary. Early indications that certain reducing agents could hinderthe action of pyruvate oxidase and or horseradish peroxidase led to theinvestigation of the alternative pyruvate detection system describedbelow.

[0144] Demonstration of Cycling: CBS/CBL/LDH System:

[0145] Pyruvate reacts with NADH in the presence of the enzyme lactatedehydrogenase to produce lactic acid and NAD. Pyruvate can be quantifiedby measuring the absorbance decrease at 340 nm. The molar absorptivityof NADH is about six-fold less than that of the peroxidase generatedchromogen. However, this does not effect the system of the presentinvention because CBL/CBS homocysteine cycling is able to generaterelatively large amounts of pyruvate.

[0146] The reagent system used to generate the data presented in FIGS.8, 9, 10, & 111 comprises serine, CBL and lactate dehydrogenase in HEPESbuffer (pH 7.2) as the first reagent. NADH in TRIS buffer (pH 8.5) andCBS are then added in sequence. All of the components can, however, becombined differently to form either a two or three reagent homocysteinemeasurement system. Reagent concentrations are shown in Table 7, andCobas FARA parameters are shown in Table 8. The NADH concentration wasadjusted to provide sufficient linearity while allowing for plasmasample absorption at 340 nm. TABLE 7 CBS/CBL/LDH CYCLING SYSTEM: ReagentComponents Chemical Concentration Reagent 1 HEPES, hemisodium salt 39.4mM HEPES, acid 12.6 mM Serine 0.72 mM Lactate dehydrogenase 33,000 U/LCBL 6.05 ug/L pH = 7.2 Reagent SR1 NADH 4.16 mM TRIS 50 mM pH = 8.5Reagent SR2 CBS 996 mg/L TRIS 50 mM Sodium Chloride 100 mM pyridoxalphosphate 5 uM Sample Diluent DTT 13 mM HEPES, hemisodium salt 56.7 mMHEPES, Acid 18.1 mM pH = 7.2

[0147] TABLE 8 CBS/CBL/LDH cycling system: Cobas FARA parametersParameter Setting GENERAL measurement mode ABS reaction modeP-T-I-SR1-I-SR2-A calibration mode LIN INTER reagent blank REAG/DILWavelength 340 nm temperature 37° C. ANALYSIS sample volume 30 μldiluent volume 10 μl diluent name H₂O (or reducing agent) reagent volume250 μl  incubation time 60 sec start reagent volume 15 μl (NADH) diluentname H₂O diluent volume  5 μl incubation time 10 sec start reagent 2volume 30 μl (CBS) diluent name H₂O diluent volume 10 μl temperaturedelay NA ABSORBANCE READINGS First  5 sec Number 16  Interval 30 secCALCULATION Number of steps 1 calculation step A ENDPOINT or KINETICFirst 6 Last 20  CALIBRATION Number of stds 1 STD1 25 or 50 μM Replicate2

[0148] Analysis of Aqueous Solutions:

[0149] The system performed well for the detection of homocysteine inaqueous solution. Typical absorption versus time plots are shown inFIGS. 8 and 9. These plots were linear with little or no apparent lagphase. Rates of NADH disappearance were relatively high. Moreover, thecalibration plot of homocysteine concentration versus absorbance waslinear over the range of 0-100 μM in the sample (FIG. 10). Thus, onlyone calibrator, e.g. 25 μM, together with a reagent blank was necessaryfor calibration.

[0150] Homocysteine solutions were prepared at various concentrations todemonstrate performance of the method over a homocysteine concentrationrange of 2-20 μM. Results are shown in Table 9 both in the presence andabsence of DTT. As mentioned above, DTT serves to reduce disulfide bondsin samples (eg serum, plasma, etc.). DTT does not significantlyinterfere with the lactate dehydrogenase catalyzed reaction. TABLE 9Demonstration of Linearity: CBS/CBL/LDH cycling system Reaction time = 3min. Observed value (uM) Homocysteine conc. (uM) (−) DTT (+) DTT 2 2 1.25 5.2 4.3 8 8.2 7.4 10 10.4 9.9 12.5 12.7 12.5 16.7 16.8 16.3 20 20.5 20

[0151] Analysis of Human Plasma Based Control Products:

[0152] Accurate performance of the CBS/CBL/LDH cycling system has beendemonstrated herein using human plasma based homocysteine controlproducts manufactured by the Bio-Rad Co. Various concentrations of DTThave been used, and incubation times have been varied in order toachieve optimum homocysteine recovery. Incubation times of 5-15 min withDTT concentrations of 1.6-3.2 mM gave homocysteine measurements wellwithin the ranges specified for various other methods.

[0153] Human Plasma Samples:

[0154] The homocysteine cycling assay has demonstrated good performanceusing human plasma samples with DTT as the reducing agent. Results agreewell with those obtained by an independent laboratory using the AbbottIMx homocysteine method (FIG. 11). Cobas FARA parameters used in thisexperiment are shown in Table 10 and reagent components are shown inTable 7. In this assay an aqueous homocysteine calibrator (25 uM) wasused. Absorbance vs. time plots for aqueous based samples were linear inthe absence of DTT and nearly linear in the presence of DTT. However,plots obtained using plasma samples were different, i.e., not linearafter the first three minutes. Under the conditions outlined in Table 7and Table 10, cycling appears to gradually slow down over time.Nevertheless, the correlation study presented in FIG. 11 demonstratesrelatively close agreement between the present method (outlined in Table7 and Table 10) with those obtained by IMx when testing human serumsamples. TABLE 10 CBS/CBL/LDH cycling system: Cobas FARA parameters forhuman plasma correlation study Parameter Setting GENERAL measurementmode ABS reaction mode P-T-I-SR1-I-SR2-A Calibration mode LIN INTERreagent blank REAG/DIL Wavelength 340 nm Temperature 37° C. ANALYSISsample volume 30 μl diluent volume 10 μl diluent name H₂O (or reducingagent) Temperature delay 900 sec  reagent volume 250 μl  Incubation time60 sec start reagent volume 15 μl (NADH) diluent name H₂O diluent volume 5 μl Incubation time 10 sec start reagent 2 volume 30 μl (CBS) diluentname H₂O diluent volume 10 μl Temperature delay NA ABSORBANCE READINGSFirst  5 sec Number 24  Interval 30 sec CALCULATION number of steps 1Calculation step A ENDPOINT or KINETIC First 1 Last 6 CALIBRATION numberof stds 1 STD1 25 or 50 μM Replicate 2

[0155] In a preferred embodiment, the rates were measured in the firstthree minutes after reaction initiation. Additional optimization of thesystem, including variation in salt and detergent concentrations, willlikely result in more consistent performance with aqueous and plasmasamples. The assay has been equally accurate when components werecombined into two or three mixtures of reagents.

EXAMPLE 7 Comparison of Homocysteine Assays

[0156] In this example, a preferred homocysteine assay in accordancewith the invention was tested and compared with two existing assays,namely a conventional HPLC technique and the commercially available IMxassay.

[0157] The assay of the invention was carried out using three reagentsas shown in Table 11. The first reagent R1 was initially in the form ofpowder and was reconstituted to a level of 1.5 g powder per 100 mLwater. The overall assay kit also included calibrators containingL-cystathionine spiked at various levels in a human plasma matrix, aswell as two control samples made up in a processed human plasma matrix.TABLE 11 CBS/CBL/LDH CYCLING SYSTEM: Reagent Components ChemicalConcentration Reagent 1 HEPES, hemisodium salt 43.3 mM Hepes, acid 12.7mM Serine 1.295 mM Lactate dehydrogenase >800 U/L NADH 1.06 mM TritonX-100 0.05% v/v Sodium azide 7.7 mM pH = 8.0 Reagent SR1 DTE 6.75 mMCitric acid 20 mM pH = 2.0 Reagent SR2 CBS 18.7 KU/L CBL 8.9 KU/LSorbitol 1.65 M Sodium chloride 100 mM Pyrdoxal phosphate 5 uM Sodiumazide 7.7 mM TRIS/HCl 50 mM pH = 8.0

[0158] TABLE 12 CBS/CBL/LDH Cycling System: Cobas MIRA ParametersParameter Setting GENERAL Measurement Mode Absorb Reaction ModeR-S-SR1-SR2 Calibration Mode Lin Regre Reagent Blank Reag/Dil Cleaner NoWavelength 340 nm Decimal Position 2 Unit umol/L ANALYSIS Post Dil.Factor No Conc. Factor No Sample cycle 1 volume 30.0 uL Diluent name H2Ovolume 25.0 uL Reagent cycle 1 volume 90 uL Start R1 cycle 2 volume 25ul Diluent name H2O volume 25 uL Start R2 cycle 17 volume 35 uL Diuentname H2O volume 15 uL CALCULATION Sample Limit No Reac. DirectionDecrease Check Off Antigen Excess No Covers. Factor 1.00 Offset 0.00Test range Low On High On Normal range Low No High No Number of Steps 1Calc. Step A Kinetic Readings First 18 Last 32 Reaction Limit NoCALIBRATION Calib. Interval Each Run Blank Reag. Range Low No High NoBlank Range Low No High No Standard Position 1 1 3.5 umol/L 2 7.0 umol/L3 17 umol/L 4 27 umol/L 5 47 umol/L 6 No 7 No Replicate Single DeviationCorrection Std. No Control CS1 position No CS2 position No CS3 positionNo

[0159] In performing this assay, the commercially available Cobas MIRAinstrument was used.

[0160] The Cobas MIRA software allows for 50 programable, 25 secondcycles. In particular, the instrument was programmed according to theparameters in Table 12. In cycle 1, 30 μL of the sample was added to acuvette, followed by 25 μL of flush water and 90 μL of liquid reagentR1. At the beginning of cycle 2, 25 μL of reagent SR1 was added with anadditional 25 μL of flush water. Thereupon, the mixture was allowed toincubate for 15 cycles (6.25 min.) to allow the reductant to liberateany bound homocysteine and to destroy any endogenous pyruvate present.At this point, 35 μL of the CBS/CBL enzyme reagent was added with anadditional 15 μL of flush water. The decrease in absorbance at 340 nmwas measured over time between cycles 18 and 32 as a measure ofhomocysteine in the sample. The total assay time between sample additionand the final absorbance reading was 32 cycles, or 13 min. and 20 sec.Since multiple samples are run in parallel, additional sample resultsare produced by the MIRA at a rate of approximately one every 30-90seconds depending on the length of the run and the number of instrumentcalibrators used. Using similar programming parameters, this assay maybe adapted to even faster instruments (eg. Roche INTEGRA, Hitachi, ACE,etc) which could yield even greater throughput rates.

[0161] A total of 24 samples were tested using the assay of theinvention and the comparative known assays. Table 13 below sets forththe homocysteine concentrations obtained using these three methods.FIGS. 12-14 are respective correlation graphs depicting graphically theresults of Table 13. As can be seen, the method of the inventioncorrelates closely with both IMx and HPLC; with closer agreementtypically seen with IMx. However, the present method is substantiallyless expensive, can be run faster, and can be performed using generallyavailable chemical analyzers.

[0162] The invention exhibits very good between-run precision as shownin Table 14. Typical within-run CV's were found to be less than 4% overhomocysteine concentrations of about 7-20 μmol/L. TABLE 13 Comparison ofHomocysteine Assays Homocysteine Concentration [μM] Sample # HPLCInvention IMx 1 9.5 11.5 11.7 2 14.3 13.1 12.1 3 8.8 7.8 7.5 4 10.6 10.19.4 5 10.5 10.1 9.9 6 8.6 9.2 7.2 7 7.6 5.8 6.2 8 5.2 3.5 3.1 9 9.6 9.39.7 10 7.7 8.7 7.5 11 11.2 11.1 9.4 12 10.5 8.3 8.1 13 11.9 8.4 8.3 1410.8 9.2 8.9 15 14.1 12.3 12.5 16 24.3 16.3 17.9 17 20.9 17.7 17.8 1824.2 22.7 22.1 19 33.6 26.4 26.4 20 21.0 20.7 18.9 21 22.7 19.6 18.1 2220.5 17.3 16.6 23 25.6 23.3 22.0 24 20.5 17.0 15.8 Average 15.2 13.312.8 Intercept [HPLC] 1.4 0.9 Slope [HPLC] 1.2 1.2 R [HPLC] 0.957 0.974Intercept [IMx] -0.3 0.5 Slope [IMx] 0.8 1.0 r [IMx] 0.976 0.990

[0163] TABLE 14 Between-Run Precision Homocysteine (μmol/L) Sample Run 1Run 2 Average % CV 1 16.35 16.46 16.41 0.47 2 12.13 12.58 12.36 2.58 319.45 21.6 20.53 7.41 4 16.29 16.79 16.54 2.14 5 11.13 10.01 10.57 7.496 7.33 7.56 7.45 2.18 7 6.31 7.24 6.78 9.71 8 7.13 5.54 6.34 17.75 914.87 14.5 14.69 1.78 10 13.81 13.41 13.61 2.08 11 26.86 28.66 27.764.58 12 12.45 13.05 12.75 3.33 13 10.15 10.74 10.45 3.99 14 9.6 10.3910.00 5.59 15 12.53 13.67 13.10 6.15 16 14.60 15.20 14.90 2.85 17 25.0325.65 25.34 1.73 18 20.77 20.03 20.40 2.56 19 31.83 32.10 31.97 0.60 2036.86 36.43 36.65 0.83 21 11.65 11.48 11.57 1.04 22 40.22 38.25 39.243.55 23 31.68 32.62 32.15 2.07 24 7.68 6.38 7.03 13.08 Average CV (%) =4.22

EXAMPLE 8 Effect of Reagent Concentration Variations

[0164] In this example, the preferred assay of the invention describedin Example 7 was performed with variations in some of the reagentcomponents, namely LDH, NADH and serine. This was done in order todetermine the optimum amounts for these reagent components. The assayswere performed as described above, with the respective components variedin concentration. Table 15 sets forth the results from these tests.TABLE 15 Effect of Reagent Component Concentration Variations Target[LDH] (U/L) Homocysteine Final range of Reaction SLOPE INTERCEPT BLANKcontrol sample: Mixture (mA/min/uM) mA/min mA/min 25-35 μM 114.5 1.21−0.14 −10.2 25.8 229.3 1.14 0.12 −11.2 28.1 458.7 1.17 0.14 −11.3 26.6917.5 0.88 1.89 −11.3 27.2 [NADH] (mM) 0.130 0.77 4.23 −10.7 32.9 0.2591.12 0.05 −11 25.2 0.389 1.09 0.45 −10.7 26.8 0.519 1.13 0.49 −11.1 26.0[SERINE] (mM) 0.125 0.64 2.23 −6.7 30.9 0.25 0.92 1.26 −9.0 28.1 0.51.13 0.63 −10.8 27.1 1 1.16 0.15 −13.6 28.3 2 1.23 0.33 −15.4 28.2

[0165] The results shown in Table 15 enabled the selection of finalreagent component levels which provided adequate sensitivity with aminimal blank rate while also trying to minimize reagent costs.

EXAMPLE 9 Effect of R1 Formulation Change on the Cycling Assay

[0166] This example determined the effect of altering the pH by changingthe buffer to TRIS, 250 mM at a pH of 8.4 and by adding lipase andα-cyclodextrin to Reagent 1. The performance of the assay was thentested after making these formulation changes. Table 16 details thereagent components used for these experiments. TABLE 16 CBS/CBL/LDHCYCLING SYSTEM: Reagent Components Chemical Concentration Reagent 1 TRIS250 mM α-cyclodextrin 0.1% Lipase ≧200 kU/L Serine 1.295 mM Lactatedehydrogenase >800 U/L NADH 1.06 mM Triton X-100 0.05% v/v Sodium azide7.7 mM pH = 8.2 Reagent SR1 TCEP 26.3 mM Reagent SR2 CBS 28.5 KU/L CBL9.4 KU/L Glycerol 10% Sorbitol 1% Pyridoxal phosphate 5 μM Sodium azide7.7 mM Potassium phosphate 100 mM pH = 7.6

[0167] By changing the buffer of the Reagent 1 to TRIS, 250 mM at pH8.4, improved assay consistency. This was because CBS and CBL aresubstantially more active at pH 8.4 and the rate of cycling is increasedby about a factor of three.

[0168] Lipase and α-cyclodextrin were added to combat the problemsassociated with turbid (lipemic) samples which are sometimes encounteredin the clinical laboratory. Such samples may arise when the patient fromwhom the sample is drawn has eaten shortly before the blood sample isdrawn or when the patient has certain disease processes going on.Turbidity can interfere with results of this testing by producing such ahigh absorbance at 340 nm that the instrument cannot accurately measurethe small changes in absorbance produced by the conversion of NADH toNAD. Turbidity can also interfere with the results of this testingdynamically by changing over time during the time when the NADH to NADconversion is measured. For example, if the amount of turbidity isdecreasing, the absorbance at 340 nm also decreases because lightscattering is less. This effect adds to the absorbance decrease causedby the reduction of pyruvate and the resulting homocysteinedetermination may be substantially too high. The addition of lipase andα-cyclodextrin to R1 did decrease the turbidity of samples, presumablyby the hydrolysis of triglycerides to glycerol and free fatty acids bylipase and by the free fatty acids forming complexes with theα-cyclodextrin. Thereby, turbid reaction mixtures clear by the time thatSR2 (R3) is added allowing for accurate measurement of the rate ofpyruvate production. Absorbances at 340 nm of many turbid reactionmixtures did rapidly decrease and level off in several minutes.Homocysteine results were accurate. However, the clearing of some turbidsamples was not complete before the addition of SR2 (R3) and therefore,the results for some samples were somewhat inaccurate. Nonetheless, theuse of lipase and alpha-cyclodextrin appears to be a very promisingapproach for achieving accurate results for turbid samples.

[0169] In addition to the changes made in the reagent constituents, thevolumes were also varied in order to assess their effects on theperformance of the assay. These changes are reflected in Table 17, whichshows a typical cycling parameter on the Cobas MIRA. Following thesechanges a correlation study was carried out on 86 human samples with theresults shown in Table 18. TABLE 17 CBS/CBL/LDH Cycling System: CobasMIRA Parameters Parameter Setting GENERAL Measurement Mode AbsorbReaction Mode R-S-SR1-SR2 Calibration Mode Linear Regression ReagentBlank Reag/Sol Cleaner No Wavelength 340 nm Decimal Position 2 Unitμmol/L ANALYSIS Post Dil. Factor No Conc. Factor No Sample cycle 1volume 20.0 μL Diluent name H₂O volume 45.0 μL Reagent cycle 1 volume 90μL Start R1 cycle 2 volume 21 μL Diluent name H₂O volume 26 μL Start R2cycle 17 volume 13 μL Diluent name H₂O volume 10 μL CALCULATION SampleLimit No Reac. Direction Decrease Check Off Antigen Excess No Covers.Factor 1.00 Offset 0.00 Test range Low On High On Normal range Low NoHigh No Number of Steps 1 Calc. Step A Kinetic Readings First 20 Last 31Reaction Limit No CALIBRATION Calib. Interval Each Run Blank Reag. RangeLow No High No Blank Range Low No High No Standard Position 1 1 0.0μmol/L 2 8.5 μmol/L 3 17.2 μmol/L 4 25.2 μmol/L 5 43.0 μmol/L 6 No 7 NoReplicate Duplicate Deviation Correction Std. No Control CS1 position NoCS2 position No CS3 position No

[0170] TABLE 18 Correlation Data with Improvement listed in Tables 16and 17. IMx Catch Sample (μmol/L) (μmol/L) 2-01 11.0 12.0 2-02 17.7 17.32-03 18.6 18.4 2-04 22.1 21.2 2-05 12.2 13.8 2-06 15.2 14.9 2-07 17.620.4 2-09 51.8 49.9 2-11 29.1 28.2 2-13 19.6 19.2 2-15 25.0 24.7 2-1622.9 23.7 2-17 21.9 21.3 2-18 25.0 23.80 2-19 22.2 22.10 2-21 23.6 20.802-22 13.0 13.60 2-23 23.1 21.80 2-24 17.0 17.60 2-26 15.9 14.80 2-27 8.89.30 2-28 9.1 9.40 2-29 5.2 5.40 2-30 8.2 8.10 2-31 4.7 5.40 2-32 6.66.80 2-33 7.8 8.10 2-34 7.0 7.0 2-35 6.9 7.3 2-36 4.8 5.7 4-01 7.2 7.34-02 7.3 9.1 4-03 6.2 5.8 4-04 9.5 9.5 4-05 6.2 7.4 4-06 8.2 8.0 4-076.8 6.7 4-08 8.1 7.4 4-09 6.5 7.9 4-10 7.5 8.5 4-11 8.1 8.6 4-12 6.1 5.74-13 6.19 6.20 4-14 5.31 5.90 4-15 7.17 6.80 4-16 5.05 7.00 4-17 5.937.80 4-18 8.57 8.10 4-19 4.87 5.70 4-21 19.37 19.50 4-22 8.61 9.30 4-2311.02 12.50 4-24 12.75 13.30 4-25 12.28 12.00 4-26 14.63 14.30 4-27 9.3311.30 4-29 18.39 19.60 4-30 14.13 14.20 4-31 8.63 8.60 4-32 9.95 9.804-33 8.08 10.20 4-34 12.34 12.00 4-35 12.16 13.10 4-36 15.75 16.30 4-3714.37 14.30 4-38 14.72 15.20 4-39 10.26 11.20 4-40 12.26 14.00 4-4124.62 25.00 4-43 27.25 29.50 4-46 21.26 19.60 4-47 22.08 23.30 4-5015.14 15.50 El 4.74 4.90 E2 8.24 8.00 E3 5.10 5.30 E6 13.79 14.50 E88.79 10.00 E9 10.57 11.50 Ell 7.10 7.40 E13 7.65 7.30 E14 12.19 12.30E15 6.47 6.14 E16 5.28 4.70 E20 7.93 7.80 Av. 12.6 12.9 Slope 0.957Intercept 0.8135 R 0.9927

[0171] As shown by this comparison table, the results of this assay withthe changed formulation was very accurate when compared with thecommercially available IMx assay.

EXAMPLE 10 Use of Other Detergents and Their Concentration Variation

[0172] In this example, different detergents were used in combinationwith EDTA as components of R1. EDTA is known to stabilize lipidparticles. Again the goal was to achieve accurate results when samplesare turbid. In particular, the detergents Brij-35 and Genapol X-80 werestudied. It was found that EDTA was particularly effective at theconcentration of 2.5 mM to help minimize non-specific decrease inabsorbance due to turbidity changes when lipemic samples are analyzed.Detergent concentrations were also varied as shown in Tables 19 and 20.One preferred composition for reagent R1 is shown in Table 21. Brij 35was present at the level of 0.025% v/v and EDTA was present at 2.5 mM.Lipase and alpha-cyclodextrin were not present because they do not seemto be necessary to achieve accuracy with turbid samples when EDTA ispresent. Brij-35 (0.025%) was also put into SR2 (R3) in the place ofTriton X-100. This may be a promising substitution because Triton X-100is considered to be an environmental hazard by some countries. Table 22shows the results of correlation study performed with the preferredreagent components. TABLE 19 Varying Genapol X-80 Concentration Results(μmol/L) change of Genapol X-80 I M × concentration in R1 Samples(μmol/L) 0.1% 0.3% 0.5% Low 7.00 6.31 6.19 5.64 Medium 12.50 11.96 11.4810.37 High 25.00 23.08 22.96 22.86 D910 14.30 14.38 14.50 14.11 D9129.40 9.41 10.60 9.50 D913 5.00 5.58 6.47 4.53 D915 21.20 23.85 21.9320.92 PBI 27 7.40 7.69 7.70 6.82 PBI 28 6.80 7.33 7.19 5.96 PBI 29 7.107.83 7.50 6.85 PBI 10 8.90 9.61 9.07 7.67 MAS 1 5.90 4.63 4.45 4.15 MAS2 14.50 14.46 15.33 14.47 MAS 3 23.10 22.60 23.07 23.65 D913X2 2.50 2.452.46 2.17 Average 11.37 11.41 11.39 10.64

[0173] TABLE 20 Varying Brij-35 Concentration in R1 Catch Results(μmol/L) as a function IMx of Brij-35 Concentration in R1 Samples(μmol/L) 0.025% 0.05% 0.1% 0.5% 1 5.88 5.03 5.35 5.26 5.09 2 14.57 15.4616.20 15.92 15.60 3 23.52 24.66 24.89 23.67 24.02 2 6.49 6.80 7.04 8.4410.74 18 10.15 10.72 11.29 9.95 10.42 21 4.62 4.86 5.84 5.73 4.96 328.90 8.17 8.38 8.67 8.43 Average 10.59 10.81 11.28 11.09 11.32

[0174] TABLE 21 CBS/CBL/LDH CYCLING SYSTEM: Reagent Components ChemicalConcentration Reagent 1 TRIS 250 mM EDTA 2.5 mM Serine 1.295 mM Lactatedehydrogenase >800 U/L NADH 1.06 mM Brij 35 0.025% w/v Sodium azide 7.7mM pH = 8.2 Reagent SR1 TCEP 26.3 mM Reagent SR2 CBS 28.5 KU/L CBL 9.4KU/L Glycerol 10% Sorbitol 1% Pyridoxal phosphate 5 μM Sodium azide 7.7mM Potassium phosphate 100 mM pH = 7.6

[0175] TABLE 22 Correlation Data with best improvement IMx Catch Sample(μmol/L)L) (μmol/ 1 5.90 5.15 2 14.57 15.36 3 23.52 23.52 701 17.9517.03 702 11.51 11.55 703 11.81 12.97 704 10.13 10.64 705 22.37 21.87706 16.97 16.79 707 7.76 7.76 708 9.35 9.90 709 7.33 6.60 710 7.24 6.90711 8.50 8.54 712 19.93 19.00 713 6.82 6.04 715 6.91 6.93 716 7.35 7.05717 9.44 9.26 718 14.16 15.61 719 5.73 5.25 720 6.32 6.00 721 11.3012.75 722 16.23 17.55 723 11.77 12.11 724 8.39 9.72 725 10.17 11.48 7266.69 6.14 727 8.01 7.50 728 10.45 10.64 729 11.73 11.27 730 14.39 14.87731 8.61 8.76 732 7.56 7.67 733 7.35 7.56 734 6.54 8.34 735 8.39 7.91Av. 10.79 10.92 slope 1.0035 intercept 0.0195 R 0.9873

[0176] When Brij-35 and EDTA were substituted for Triton X-100 andlipase plus α-cyclodextrin, there was some clearing of the turbidreaction mixtures. However, perhaps not as much as in the case whenlipase and alpha-cyclodextrin are also used. Importantly, theabsorbances at 340 nm quickly stabilized with zero rates of change. Thuswhen SR2 (R3) was added, the rate of pyruvate production is accuratelymeasured and accurate results are obtained even in the cases of samplesappearing like milk. This is even the case when the time betweenaddition of R1 and SR2 (R3) is shortened by 2-4 minutes, which may benecessary to apply the homocysteine assay of the present invention toother automated instruments.

EXAMPLE 11

[0177] Results from Changing Reducing Agent to TCEP and Alterations inConcentration of TCEP

[0178] For this example, the reducing agent DTE was replaced with Tris(2-carboxyethylphosphine) hydrochloride (TCEP). TCEP has the advantageof being stable in purified water for an extended period of time.Replacement of DTE with TCEP reduced the reagent blank reaction fromabout 12 to 15 mA/min to about 4 to 5 mA/min. The lower reagent blankenables better Homocysteine assay precision. To find the optimumconcentration of TCEP for this Homocysteine assay, a range ofconcentration was tested. The results from these experiments are givenin Table 23. TABLE 23 Varying TCEP Concentration in SR1 HomocysteineResults (μmol/L) SR1 Catch Assay Formulation IMx A B C D E F MAS 1 5.886.37 6.18 5.48 5.51 4.42 4.17 MAS 2 14.57 16.49 15.67 15.95 16.03 15.8014.77 MAS 3 23.52 23.19 21.98 23.33 25.11 24.02 23.09 PBI 20 7.2 5.816.24 7.81 8.00 6.95 8.33 PBI 22 7.8 7.61 7.66 8.28 8.69 8.44 8.23 PBI 365.1 4.16 4.61 4.71 5.05 5.18 4.71 PBI 39 4.6 3.11 4.25 4.38 4.47 3.684.09 Doreen 709 7.33 6.68 6.64 6.71 7.28 6.23 6.43 Average 9.50 9.179.15 9.58 10.02 9.34 9.22

EXAMPLE 12

[0179] Determination of the Effect of Changing the R3 Buffer toPhosphate and the Addition of Glycerol to the Buffer

[0180] In order to have enzyme mixtures that would have longer stabilityand assay compatibility, the enzyme formulation was changed to a mixturethat provides significantly longer shelf-life than the previousformulation. The significant changes involved replacement of Tris bufferwith a phosphate and addition of glycerol to the buffer. Potassiumphosphate at concentrations ranging from 50 mM to 500 mM at pH 7.6 wereused and glycerol concentrations ranging from 0.5% to 15.0% (v/v) wereused. As a result from these changes, the shelf life was increasedsignificantly from weeks to more than a year at 4° C. In preferredforms, potassium phosphate can range from about 50 mM to about 250 mM.More preferably, potassium phosphate can range from about 75 mM to about150 mM. A particularly effective concentration of potassium phosphate inthis experiment was 100 mM. Glycerol can range from about 1-15%. Morepreferably, this range is from about 5-13%. A particularly effectiveconcentration of glycerol in this experiment was 10%.

EXAMPLE 13 Results Obtained Using Different Instruments

[0181] To determine the overall improvement resulting from all thechanges to the assay, precision evaluations were done on severalinstruments by writing protocol that is specific to that instrument.Table 24 sets forth the parameters and settings used in accordance withthe present invention with the Roche Hitachi 911 and Table 25 sets forththe same parameters and settings when the Beckman CX-5 is used. Beloware the tables showing the results of these precision tests. Theseresults are significantly better than what was obtained with the earlierformulations of the assay. On the Hitachi 911 (Table 26) and Beckman CX5(Table 27) instruments, each sample shown was run 20× and the within runprecision was determined. Percent C. V. for samples as low as 5.92μmol/L was 3.31% and samples in the range of 23.01 μmol/L to 25.4 μmol/Lwas 1.9% to 2.03%. A total precision (Table 28) of between 7.3% and 2.6%was obtained on samples with homocysteine values of 4.6 μmol/L and 27.0μmol/L on the Cobas MIRA Plus. Each sample was run in duplicate, 2× perday for a total of 20 days. TABLE 24 Catch Homocysteine MethodParameters for the Roche Hitachi 911 Test THCY Assay Code Rate-A, 15Wavelength (2nd/Primary) 376/340 Assay Point 35-49-0-0 Serum S. Vol.(Normal) 28 S. Vol. (Decrease) 20 S. Vol. (Increase) 30 Urine S. Vol.(Normal) 10 S. Vol. (Decrease) 10 S. Vol. (Increase) 10 ABS. Limit0-0-Decrease Prozone Limit 0-0-Lower Reagent T1 200 μL - 0-0-00322-0 T225 μL - 0-0-00322-0 T3 0 - 0-0-00322-0 Calibration Type Linear-2-2-0Auto Time Out Blank 0 Span 0 2 Point 0 Full 0 Auto Change Lot CancelBottle Cancel Select Tests via Keyboard: ENTER SD Limit 100 DuplicateLimit 1000 Sensitivity Limit 0 S1 ABS Limit (−32000) (32000) CompensatedLimit Blank PARAMETER PAGE 2 Test THCY - 00322 Test Name THCY Unit:μmol/L Data Mode On Board Report Name Homocysteine Control Interval 0Instrument Factor (Y = aX + b) a 1 b 0 Expected Values user definedTechnical Limit Serum 0-10000 Urine (−99999) (999999) STDConc.-Pos.-Sample-Pre.-Dil.-Calib. 1 0.0-18-28-0-0-501 225.2-26-28-0-0-026 3 0 4 0 5 0 6 0

[0182] For this experiment, T1 is prepared by mixing R1 and deionizedwater in the ratio of 9 to 8.1. Reagents T2 and T4 correspond toReagents SR1 and SR2, respectively. TABLE 25 Catch Homocysteine MethodParameters for the Beckman CX-5 Chemistry Name: HOMOCYSTEINE Test NameHOMO Calculation Factor: 0 Reaction Type: RATE 1 Math Model: Linear CalTime Limit: 24 Hrs Reaction Direction: Decreasing No. of Calibrators: 2Units: μmol/L Decimal Precision X.XX Primary Wavelength: 340 nmSecondary Wavelength: 380 nm Sample Volume 25 μL Primary Inject Rgt: A179 μL B 22 μL Secondary Inject Rgt: C 14 μL Add Time: 592 secCALIBRATORS #1 0.00 #2 24.8 MULTIPOINT SPAN 1-2: −0.001 REAGENT BLANKStart Read: 100 sec End Read: 200 sec Low ABS Limit: −1.5 High ABS Limit1.5 USABLE RANGE Lower Limit: 0.00 Upper Limit: 99999.00 SUBSTRATEDEPLETION Initial Rate: −99.999 Delta ABS 1.5

[0183] For this example, Reagent A is prepared by mixing R1 anddeionized water in the ration of 9 to 8.1. Reagents B and C correspondto MIRA reagents SR1 and SR2. TABLE 26 Within Run Precision on Hitachi911 Within Run Low Std. Dev. 0.224 Mean = 8.3 μmol/L % C.V. 2.17 MediumStd. Dev. 0.324 Mean = 11.9 μmol/L % C. V. 2.71 High Std. Dev. 0.516Mean = 25.4 μmol/L % C. V. 2.03

[0184] TABLE 27 Within Run Precision on Beckman CX5 Within Run Low Std.Dev. 0.196 Mean = 5.92 μmol/L % C.V. 3.31 Medium Std. Dev. 0.195 Mean =11.12 μmol/L % C. V. 1.70 High Std. Dev. 0.437 Mean = 23.01 μmol/L % C.V. 1.90

[0185] TABLE 28 Total Precision on Roche MIRA-Plus Within BetweenBetween Run Run Day Total Low Std. 0.31 0.14 0.04 0.34 Mean = 4.6 μmol/LDev. % C.V. 6.6 3.1 0.8 7.3 Medium Std. 0.40 0.08 0.23 0.47 Mean = 11.6μmol/L Dev. % C.V. 3.5 0.7 2.0 4.0 High Std. 0.44 0.39 0.39 0.71 Mean =27.0 μmol/L Dev. % C.V. 1.6 1.5 1.4 2.6

EXAMPLE 14 Adaptability of the Assay to a 2-Reagent System

[0186] The present invention can also be adapted to system which usesonly two reagents. This makes the invention adaptable to wider range ofinstruments including those that can only use two reagents. Table 29shows the reagent contents of the two-reagent system and Table 30 showsthe MIRA plus parameters of how the assay was performed on thatchemistry analyzer. Shown in Table 31 are the comparison data obtainedusing the 3-reagents system versus the 2-reagents system. TABLE 29CBS/CBL/LDH CYCLING SYSTEM: Reagent Components for 2-Reagents ChemicalConcentration Reagent 1 TRIS 250 mM EDTA 2.5 mM Serine 1.295 mM Lactatedehydrogenase >800 U/L NADH 1.06 mM Brij 35 0.025% v/v Sodium azide 7.7mM TCEP 4.05 mM pH = 8.2 Reagent SR1 CBS 28.5 KU/L CBL 9.4 KU/L Glycerol10% Sorbitol 1% Pyrdoxal phosphate 5 μM Sodium azide 7.7 mM Potassiumphosphate 100 mM pH = 7.6

[0187] TABLE 30 CBS/CBL/LDH Cycling System for 2-Reagents: Cobas MIRAParameters Parameter Setting GENERAL Measurement Mode Absorb ReactionMode R-S-SR1 Calibration Mode Linear Regression Reagent Blank Reag/SolCleaner No Wavelength 340 nm Decimal Position 2 Unit μmol/L ANALYSISPost Dil. Factor No Conc. Factor No Sample cycle 1 volume 20.0 μLDiluent name H₂O volume 45.0 μL Reagent cycle 1 volume 137 μL Start R1cycle 16 volume 13 μL Diuent name H₂O volume 10 μL CALCULATION SampleLimit No Reac. Direction Decrease Check Off Antigen Excess No Covers.Factor 1.00 Offset 0.00 Test range Low On High On Normal range Low NoHigh No Number of Steps 1 Calc. Step A Kinetic Readings First 19 Last 30Reaction Limit No CALIBRATION Calib. Interval Each Run Blank Sol-Pos 1Reag. Range Low No High No Blank Range Low No High No Standard Position1 1 0.0 μmol/L 2 8.5 μmol/L 3 17.2 μmol/L 4 25.2 μmol/L 5 43.0 μmol/L 6No 7 No Replicate Duplicate Deviation Correction Std. No Control CS1position No CS2 position No CS3 position No

[0188] TABLE 31 Comparison of 2-Reagent System versus 3-Reagent System.Catch Inc. Hcy Assay (μmol/L) IMx 2-Reagent 3-Reagent Samples (μmol/L)System System MAS 1 5.88 5.55 5.17 MAS 2 14.57 16.14 15.70 MAS 3 23.5223.99 23.47 P 10 8.90 9.32 9.65 P 11 7.60 7.45 7.27 P 33 7.80 8.11 8.29P 34 5.93 5.21 5.69 754 + 848 16.00 15.68 16.15 Average 11.28 11.4311.42

EXAMPLE 15 Adaptability of the Present Invention for Use withSingle-Point Calibration

[0189] The homocysteine assay of the present invention has also beenadapted to using a single calibration point, with the zero calibratorserving as the blank. Table 32 below shows the parameters for performinga single point calibration on the Mira instrument. The data below (Table33) shows that using single point calibration in the slope average modegenerates results that are substantially equivalent to using themulti-point calibration curve. TABLE 32 CBS/CBL/LDH Cycling System:Cobas MIRA Parameters for Single Point Calibration Parameter SettingGENERAL Measurement Mode Absorb Reaction Mode R-S-SR1 Calibration ModeSlope Average Reagent Blank Reag/Sol Cleaner No Wavelength 340 nmDecimal Position 2 Unit μmol/L ANALYSIS Post Dil. Factor No Conc. FactorNo Sample cycle 1 volume 20.0 μL Diluent name H₂O volume 45.0 μL Reagentcycle 1 volume 137 μL Start R1 cycle 16 volume 13 μL Diuent name H₂Ovolume 10 μL CALCULATION Sample Limit No Reac. Direction Decrease CheckOff Antigen Excess No Covers. Factor 1.00 Offset 0.00 Test range Low OnHigh On Normal range Low No High No Number of Steps 1 Calc. Step AKinetic Readings First 19 Last 30 Reaction Limit No CALIBRATION Calib.Interval Each Run Blank Sol-Pos 1 Reag. Range Low No High No Blank RangeLow No High No Standard Position 4 1 25.2 μmol/L 2 NO 3 NO ReplicateDuplicate Deviation Correction Std. No Control CS1 position No CS2position No CS3 position No

[0190] TABLE 33 Single-Point versus Multi-Point Calibration Catch Inc.Hcy Assay (μmol/L) IMx Single Point Multi-Point Samples (μmol/L)Calibration Calibration MAS 1 5.88 5.03 5.17 MAS 2 14.57 15.18 15.70 MAS3 23.52 22.97 23.47 PB1 10 8.90 8.82 9.65 PBI 11 7.60 8.26 7.27 PBI 337.80 8.05 8.29 PBI 34 5.93 5.80 5.69 754 + 848 16.00 15.17 16.15 Average11.28 11.16 11.42

EXAMPLE 16 Study of Homocysteine Assays Described in WO 00/28071

[0191] PCT Publication WO 00/28071 describes in detail two homocysteineassays. In the “first embodiment”, the assay is performed without areduction step. To convert that method to one useful for human plasmahomocysteine measurements, either a separate off-line or a homogeneousreduction step must be incorporated. Publication WO 00/28071 describes aseparate off-line reduction step perhaps because a pyruvate oxidasecycling system is used. That step entails an incubation of 20 min.before the reduced specimen can be analyzed, which greatly increasestotal assay time and labor cost. Moreover, if the sample is to beassayed for more than one analyte in the clinical laboratory, theprimary serum or plasma sample must be split into two aliquots, one forthe homocysteine test and one for any other ordered laboratory test. Incontrast, the present invention enables homogeneous reduction since aLDH cycling system is used (Example 7).

[0192] Here, in Example 16 (Table 34), it is shown that DTT in fact doesinterfere with the pyruvate oxidase detection system described in the“second embodiment” of WO 00/28071. In particular, the reagent and DTTcompositions described in the “second embodiment” were used, and assayswith and without DTT were carried out against known pyruvate standards(12.5, 25, and 50 μmol/L) using a Cobas FARA instrument. In the case ofthe DTT test, 40 μL of each standard was mixed with 30 μL of the DTTsolution, followed by incubation for 100 sec. Thereafter, 90 μL ofpyruvate oxidase reagent was added and incubated for 30 sec. Absorbancereadings were then taken at 30, 60, 90, 120, 150, 180 and 210 sec. Inthe “no DTT” test, the same procedure was followed except that no DTTwas added. Table 34 sets forth the results of this experiment using the210 sec. readings. FIGS. 15 and 16 further illustrate the results ofthis test. TABLE 34 Effect of DTT on PO/HRP pyruvate detection systemWITHOUT DTT WITH DTT Blank 0.0021 −0.0021 Factor 2212 −11765   50 μmol/LStandard 51.5 35.3   25 μmol/L Standard 24.1 0.0 12.5 μmol/L Standard9.7 −21.2 Delta change at 30 sec. (50 μmol std.) 0.0643 −0.0062 Deltachange at 90 sec. (50 μmol std.) 0.0890 −0.0103 Delta change at 120 sec.(50 μmol std.) 0.0903 −0.0113 Delta change at 210 sec. (50 μmol std.)0.0906 −0.0113

[0193] These data clearly demonstrate the necessity of the separatereduction step described in the “second embodiment” of WO 00/28071. Thepresence of the reductant interferes with the pyruvate oxidase detectionsystem used to assay for pyruvate. Therefore, the data presented hereconfirm that the time-consuming separate off-line reduction step isessential in the “second embodiment.” In addition, the off-linereduction step described in WO 00/28071 will likely elevatesignificantly the assay complexity and cost relative to the presentinvention.

[0194] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims. The scope ofthe invention should, therefore, be determined not with reference to theabove description, but instead should be determined with reference tothe appended claims along with their full scope of equivalents.

[0195] All publications and patent documents cited in this applicationare incorporated by reference in their entirety for all purposes to thesame extent as if each individual publication or patent document were soindividually denoted.

1 21 1 30 DNA Artificial PCR Primer 1 cgacggatcc gatggcggac aaaaagcttg30 2 29 DNA Artificial Sequence PCR Primer 2 cgcagcagct gttatacaattcgcgcaaa 29 3 29 DNA Artificial Sequence PCR PRimer 3 cggggatcctgatgcatgca tgataagga 29 4 27 DNA Artificial Sequence PCR Primer 4cggcattacc gtttcactaa tttattg 27 5 25 DNA Artificial Sequence PCR Primer5 gttggatccg gcattaccgt ttcac 25 6 30 DNA Artificial PCR Primer 6ggtggatcca tcaatagata cgtacgtcct 30 7 25 DNA Artificial PCR Primer 7ttttagttta tgtatgtgtt ttttg 25 8 41 DNA Artificial PCR Primer 8aacacataca taaactaaaa atgactaaat ctgagcagca a 41 9 39 DNA Artificial PCRPrimer 9 atgatgatga tgatgatgac ctgctaagta gctcagtaa 39 10 36 DNAArtificial PCR Primer 10 catcatcatc atcatcatta aataagaacc cacgct 36 1130 DNA Artificial Consensus MetR receptor Sequence 11 gttaatgttgaacaaatctc atgttgcgtg 30 12 35 DNA Artificial PCR Primer 12 gcgggtcgactatgactaaa tctgagcagc aagcc 35 13 31 DNA Artificial PCR Primer 13gcgtgcggcc gcgttatgct aagtagctca g 31 14 1524 DNA Saccharomycescerevisiae 14 atgactaaat ctgagcagca agccgattca agacataacg ttatcgacttagttggtaac 60 accccattga tcgcactgaa aaaattgcct aaggctttgg gtatcaaaccacaaatttat 120 gctaagctgg aactatacaa tccaggtggt tccatcaaag acagaattgccaagtctatg 180 gtggaagaag ctgaagcttc cggtagaatt catccttcca gatctactctgatcgaacct 240 acttctggta acaccggtat cggtctagct ttaatcggcg ccatcaaaggttacagaact 300 atcatcacct tgccggaaaa aatgtctaac gagaaagttt ctgtcctaaaggctctgggt 360 gctgaaatca tcagaactcc aactgctgct gcctgggatt ctccagaatcacatattggt 420 gttgctaaga agttggaaaa agagattcct ggtgctgtta tacttgaccaatataacaat 480 atgatgaacc cagaagctca ttactttggt actggtcgcg aaatccaaagacagctagaa 540 gacttgaatt tatttgataa tctacgcgct gttgttgctg gtgctggtactggtgggact 600 attagcggta tttccaagta cttgaaagaa cagaatgata agatccaaatcgttggtgct 660 gagggattcg gttcaatttt agcccaacct gaaaacttga ataagactgatatcactgac 720 tacaaagttg agggtattgg ttatgatttt gttcctcagg ttttggacagaaaattaatt 780 gatgtttggt ataagacaga cgacaagcct tctttcaaat acgccagacaattgatttct 840 aacgaaggtg tcttggtggg tggttcttcc ggttctacct tcactgcggttgtgaaatac 900 tgtgaagacc accctgaact gactgaagat gatgtcattg ttgccatattcccagattcc 960 atcaggtcgt acctaaccaa attcgtcgat gacgaatggt tgaaaaagaacaatttgtgg 1020 gatgatgacg tgttggcccg ttttgactct tcaaagctgg aggcttcgacgacaaaatac 1080 gctgatgtgt ttggtaacgc tactgtaaag gatcttcact tgaaaccggttgtttccgtt 1140 aaggaaaccg ctaaggtcac tgatgttatc aagatattaa aagacaatggctttgaccaa 1200 ttgcctgtgt tgactgaaga cggcaagttg tctggtttag ttactctctctgagcttcta 1260 agaaaactat caatcaataa ttcaaacaac gacaacacta taaagggtaaatacttggac 1320 ttcaagaaat taaacaattt caatgatgtt tcctcttaca acgaaaataaatccggtaag 1380 aagaagttta ttaaattcga tgaaaactca aagctatctg acttgaatcgtttctttgaa 1440 aaaaactcat ctgccgttat cactgatggc ttgaaaccaa tccatatcgttactaagatg 1500 gatttactga gctacttagc ataa 1524 15 43 DNA Artificial PCRPrimer 15 ctatatcgta ataatgacta aatctgagca gcaagccgat tca 43 16 40 DNAArtificial PCR Primer 16 agggctcgag gatcccgggg gtgctattat gaatgcacgg 4017 35 DNA Artificial PCR Primer 17 cggggatccg acgttttgcc cgcaggcgggaaacc 35 18 39 DNA Artificial PCR Primer 18 agatttagtc attattacgatatagttaat agttgatag 39 19 428 PRT Escherichia coli 19 Met Arg Gly SerHis His His His His His Gly Met Ala Ser Met Thr 1 5 10 15 Gly Gly GlnGln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp 20 25 30 Pro Met AlaAsp Lys Lys Leu Asp Thr Gln Leu Val Asn Ala Gly Arg 35 40 45 Ser Lys LysTyr Thr Leu Gly Ala Val Asn Ser Val Ile Gln Arg Ala 50 55 60 Ser Ser LeuVal Phe Asp Ser Val Glu Ala Lys Lys His Ala Thr Arg 65 70 75 80 Asn ArgAla Asn Gly Glu Leu Phe Tyr Gly Arg Arg Gly Thr Leu Thr 85 90 95 His PheSer Leu Gln Gln Ala Met Cys Glu Leu Glu Gly Gly Ala Gly 100 105 110 CysVal Leu Phe Pro Cys Gly Ala Ala Ala Val Ala Asn Ser Ile Leu 115 120 125Ala Phe Ile Glu Gln Gly Asp His Val Leu Met Thr Asn Thr Ala Tyr 130 135140 Glu Pro Ser Gln Asp Phe Cys Ser Lys Ile Leu Ser Lys Leu Gly Val 145150 155 160 Thr Thr Ser Trp Phe Asp Pro Leu Ile Gly Ala Asp Ile Val LysHis 165 170 175 Leu Gln Pro Asn Thr Lys Ile Val Phe Leu Glu Ser Pro GlySer Ile 180 185 190 Thr Met Glu Val His Asp Val Pro Ala Ile Val Ala AlaVal Arg Ser 195 200 205 Val Val Pro Asp Ala Ile Ile Met Ile Asp Asn ThrTrp Ala Ala Gly 210 215 220 Val Leu Phe Lys Ala Leu Asp Phe Gly Ile AspVal Ser Ile Gln Ala 225 230 235 240 Ala Thr Lys Tyr Leu Val Gly His SerAsp Ala Met Ile Gly Thr Ala 245 250 255 Val Cys Asn Ala Arg Cys Trp GluGln Leu Arg Glu Asn Ala Tyr Leu 260 265 270 Met Gly Gln Met Val Asp AlaAsp Thr Ala Tyr Ile Thr Ser Arg Gly 275 280 285 Leu Arg Thr Leu Gly ValArg Leu Arg Gln His His Glu Ser Ser Leu 290 295 300 Lys Val Ala Glu TrpLeu Ala Glu His Pro Gln Val Ala Arg Val Asn 305 310 315 320 His Pro AlaLeu Pro Gly Ser Lys Gly His Glu Phe Trp Lys Arg Asp 325 330 335 Phe ThrGly Ser Ser Gly Leu Phe Ser Phe Val Leu Lys Lys Lys Leu 340 345 350 AsnAsn Glu Glu Leu Ala Asn Tyr Leu Asp Asn Phe Ser Leu Phe Ser 355 360 365Met Ala Tyr Ser Trp Gly Gly Tyr Glu Ser Leu Ile Leu Ala Asn Gln 370 375380 Pro Glu His Ile Ala Ala Ile Arg Pro Gln Gly Glu Ile Asp Phe Ser 385390 395 400 Gly Thr Leu Ile Arg Leu His Ile Gly Leu Glu Asp Val Asp AspLeu 405 410 415 Ile Ala Asp Leu Asp Ala Gly Phe Ala Arg Ile Val 420 42520 1252 PRT Saccharomyces cerevisiae 20 Met Ser Pro Ile Leu Gly Tyr TrpLys Ile Lys Gly Leu Val Gln Pro 1 5 10 15 Thr Arg Leu Leu Leu Glu TyrLeu Glu Glu Lys Tyr Glu Glu His Leu 20 25 30 Tyr Glu Arg Asp Glu Gly AspLys Trp Arg Asn Lys Lys Phe Glu Leu 35 40 45 Gly Leu Glu Phe Pro Asn LeuPro Tyr Tyr Ile Asp Gly Asp Val Lys 50 55 60 Leu Thr Gln Ser Met Ala IleIle Arg Tyr Ile Ala Asp Lys His Asn 65 70 75 80 Met Leu Gly Gly Cys ProLys Glu Arg Ala Glu Ile Ser Met Leu Glu 85 90 95 Gly Ala Val Leu Asp IleArg Tyr Gly Val Ser Arg Ile Ala Tyr Ser 100 105 110 Lys Asp Phe Glu ThrLeu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu 115 120 125 Met Leu Lys MetPhe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn 130 135 140 Gly Asp HisVal Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp 145 150 155 160 ValVal Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu 165 170 175Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr 180 185190 Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala 195200 205 Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Glu Val Leu210 215 220 Phe Gln Gly Pro Leu Gly Ser Pro Gly Ile Pro Gly Ser Thr MetThr 225 230 235 240 Lys Ser Glu Gln Gln Ala Asp Ser Arg His Asn Val IleAsp Leu Val 245 250 255 Gly Asn Thr Pro Leu Ile Ala Leu Lys Lys Leu ProLys Ala Leu Gly 260 265 270 Ile Lys Pro Gln Ile Tyr Ala Lys Leu Glu LeuTyr Asn Pro Gly Gly 275 280 285 Ser Ile Lys Asp Arg Ile Ala Lys Ser MetVal Glu Glu Ala Glu Ala 290 295 300 Ser Gly Arg Ile His Pro Ser Arg SerThr Leu Ile Glu Pro Thr Ser 305 310 315 320 Gly Asn Thr Gly Ile Gly LeuAla Leu Ile Gly Ala Ile Lys Gly Tyr 325 330 335 Arg Thr Ile Ile Thr LeuPro Glu Lys Met Ser Asn Glu Lys Val Ser 340 345 350 Val Leu Lys Ala LeuGly Ala Glu Ile Ile Arg Thr Pro Thr Ala Ala 355 360 365 Ala Trp Asp SerPro Glu Ser His Ile Gly Val Ala Lys Lys Leu Glu 370 375 380 Lys Glu IlePro Gly Ala Val Ile Leu Asp Gln Tyr Asn Asn Met Met 385 390 395 400 AsnPro Glu Ala His Tyr Phe Gly Thr Gly Arg Glu Ile Gln Arg Gln 405 410 415Leu Glu Asp Leu Asn Leu Phe Asp Asn Leu Arg Ala Val Val Ala Gly 420 425430 Ala Gly Thr Gly Gly Thr Ile Ser Gly Ile Ser Lys Tyr Leu Lys Glu 435440 445 Gln Asn Asp Lys Ile Gln Ile Val Gly Ala Asp Pro Phe Gly Ser Ile450 455 460 Leu Ala Gln Pro Glu Asn Leu Asn Lys Thr Asp Ile Thr Asp TyrLys 465 470 475 480 Val Glu Gly Ile Gly Tyr Asp Phe Val Pro Gln Val LeuAsp Arg Lys 485 490 495 Leu Ile Asp Val Trp Tyr Lys Thr Asp Asp Lys ProSer Phe Lys Tyr 500 505 510 Ala Arg Gln Leu Ile Ser Asn Glu Gly Val LeuVal Gly Gly Ser Ser 515 520 525 Gly Ser Ala Phe Thr Ala Val Val Lys TyrCys Glu Asp His Pro Glu 530 535 540 Leu Thr Glu Asp Asp Val Ile Val AlaIle Phe Pro Asp Ser Ile Arg 545 550 555 560 Ser Tyr Leu Thr Lys Phe ValAsp Asp Glu Trp Leu Lys Lys Asn Asn 565 570 575 Leu Trp Asp Asp Asp ValLeu Ala Arg Phe Asp Ser Ser Lys Leu Glu 580 585 590 Ala Ser Thr Thr LysTyr Ala Asp Val Phe Gly Asn Ala Thr Val Lys 595 600 605 Asp Leu His LeuLys Pro Val Val Ser Val Lys Glu Thr Ala Lys Val 610 615 620 Thr Asp ValIle Lys Ile Leu Lys Asp Asn Gly Phe Asp Gln Leu Pro 625 630 635 640 ValLeu Thr Glu Asp Gly Lys Leu Ser Gly Leu Val Thr Leu Ser Glu 645 650 655Leu Leu Arg Lys Leu Ser Ile Asn Asn Ser Asn Asn Asp Asn Thr Ile 660 665670 Lys Gly Lys Tyr Leu Asp Phe Lys Lys Leu Asn Asn Phe Asn Asp Val 675680 685 Ser Ser Tyr Asn Glu Asn Lys Ser Gly Lys Lys Lys Phe Ile Lys Phe690 695 700 Asp Glu Asn Ser Lys Leu Ser Asp Leu Asn Arg Phe Phe Glu LysAsn 705 710 715 720 Ser Ser Ala Val Ile Thr Asp Gly Leu Lys Pro Ile HisIle Val Thr 725 730 735 Lys Met Asp Leu Leu Ser Tyr Leu Ala Met Thr LysSer Glu Gln Gln 740 745 750 Ala Asp Ser Arg His Asn Val Ile Asp Leu ValGly Asn Thr Pro Leu 755 760 765 Ile Ala Leu Lys Lys Leu Pro Lys Ala LeuGly Ile Lys Pro Gln Ile 770 775 780 Tyr Ala Lys Leu Glu Leu Tyr Asn ProGly Gly Ser Ile Lys Asp Arg 785 790 795 800 Ile Ala Lys Ser Met Val GluGlu Ala Glu Ala Ser Gly Arg Ile His 805 810 815 Pro Ser Arg Ser Thr LeuIle Glu Pro Thr Ser Gly Asn Thr Gly Ile 820 825 830 Gly Leu Ala Leu IleGly Ala Ile Lys Gly Tyr Arg Thr Ile Ile Thr 835 840 845 Leu Pro Glu LysMet Ser Asn Glu Lys Val Ser Val Leu Lys Ala Leu 850 855 860 Gly Ala GluIle Ile Arg Thr Pro Thr Ala Ala Ala Trp Asp Ser Pro 865 870 875 880 GluSer His Ile Gly Val Ala Lys Lys Leu Glu Lys Glu Ile Pro Gly 885 890 895Ala Val Ile Leu Asp Gln Tyr Asn Asn Met Met Asn Pro Glu Ala His 900 905910 Tyr Phe Gly Thr Gly Arg Glu Ile Gln Arg Gln Leu Glu Asp Leu Asn 915920 925 Leu Phe Asp Asn Leu Arg Ala Val Val Ala Gly Ala Gly Thr Gly Gly930 935 940 Thr Ile Ser Gly Ile Ser Lys Tyr Leu Lys Glu Gln Asn Asp LysIle 945 950 955 960 Gln Ile Val Gly Ala Asp Pro Phe Gly Ser Ile Leu AlaGln Pro Glu 965 970 975 Asn Leu Asn Lys Thr Asp Ile Thr Asp Tyr Lys ValGlu Gly Ile Gly 980 985 990 Tyr Asp Phe Val Pro Gln Val Leu Asp Arg LysLeu Ile Asp Val Trp 995 1000 1005 Tyr Lys Thr Asp Asp Lys Pro Ser PheLys Tyr Ala Arg Gln Leu 1010 1015 1020 Ile Ser Asn Glu Gly Val Leu ValGly Gly Ser Ser Gly Ser Ala 1025 1030 1035 Phe Thr Ala Val Val Lys TyrCys Glu Asp His Pro Glu Leu Thr 1040 1045 1050 Glu Asp Asp Val Ile ValAla Ile Phe Pro Asp Ser Ile Arg Ser 1055 1060 1065 Tyr Leu Thr Lys PheVal Asp Asp Glu Trp Leu Lys Lys Asn Asn 1070 1075 1080 Leu Trp Asp AspAsp Val Leu Ala Arg Phe Asp Ser Ser Lys Leu 1085 1090 1095 Glu Ala SerThr Thr Lys Tyr Ala Asp Val Phe Gly Asn Ala Thr 1100 1105 1110 Val LysAsp Leu His Leu Lys Pro Val Val Ser Val Lys Glu Thr 1115 1120 1125 AlaLys Val Thr Asp Val Ile Lys Ile Leu Lys Asp Asn Gly Phe 1130 1135 1140Asp Gln Leu Pro Val Leu Thr Glu Asp Gly Lys Leu Ser Gly Leu 1145 11501155 Val Thr Leu Ser Glu Leu Leu Arg Lys Leu Ser Ile Asn Asn Ser 11601165 1170 Asn Asn Asp Asn Thr Ile Lys Gly Lys Tyr Leu Asp Phe Lys Lys1175 1180 1185 Leu Asn Asn Phe Asn Asp Val Ser Ser Tyr Asn Glu Asn LysSer 1190 1195 1200 Gly Lys Lys Lys Phe Ile Lys Phe Asp Glu Asn Ser LysLeu Ser 1205 1210 1215 Asp Leu Asn Arg Phe Phe Glu Lys Asn Ser Ser AlaVal Ile Thr 1220 1225 1230 Asp Gly Leu Lys Pro Ile His Ile Val Thr LysMet Asp Leu Leu 1235 1240 1245 Ser Tyr Leu Ala 1250 21 1188 DNAEscherichia coli 21 atggcggaca aaaagcttga tactcaactg gtgaatgcaggacgcagcaa aaaatacact 60 ctcggcgcgg taaatagcgt gattcagcgc gcttcttcgctggtctttga cagtgtagaa 120 gccaaaaaac acgcgacacg taatcgcgcc aatggagagttgttctatgg acggcgcgga 180 acgttaaccc atttctcctt acaacaagcg atgtgtgaactggaaggtgg cgcaggctgc 240 gtgctatttc cctgcggggc ggcagcggtt gctaattccattcttgcttt tatcgaacag 300 ggcgatcatg tgttgatgac caacaccgcc tatgaaccgagtcaggattt ctgtagcaaa 360 atcctcagca aactgggcgt aacgacatca tggtttgatccgctgattgg tgccgatatc 420 gttaagcatc tgcagccaaa cactaaaatc gtgtttctggaatcgccagg ctccatcacc 480 atggaagtcc acgacgttcc ggcgattgtt gccgccgtacgcagtgtggt gccggatgcc 540 atcattatga tcgacaacac ctgggcagcc ggtgtgctgtttaaggcgct ggattttggc 600 atcgatgttt ctattcaagc cgccaccaaa tatctggttgggcattcaga tgcgatgatt 660 ggcactgccg tgtgcaatgc ccgttgctgg gagcagctacgggaaaatgc ctatctgatg 720 ggccagatgg tcgatgccga taccgcctat ataaccagccgtggcctgcg cacattaggt 780 gtgcgtttgc gtcaacatca tgaaagcagt ctgaaagtggctgaatggct ggcagaacat 840 ccgcaagttg cgcgagttaa ccaccctgct ctgcctggcagtaaaggtca cgaattctgg 900 aaacgagact ttacaggcag cagcgggcta ttttcctttgtgcttaagaa aaaactcaat 960 aatgaagagc tggcgaacta tctggataac ttcagtttattcagcatggc ctactcgtgg 1020 ggcgggtatg aatcgttgat cctggcaaat caaccagaacatatcgccgc cattcgccca 1080 caaggcgaga tcgattttag cgggaccttg attcgcctgcatattggtct ggaagatgtc 1140 gacgatctga ttgccgatct ggacgccggt tttgcgcgaattgtataa 1188

We claim:
 1. The method of determining the amount of homocysteine in asample comprising the steps of: (a) contacting said sample with anenzyme and a substrate, said enzyme operable to convert homocysteine tocystathionine in the presence of said substrate; (b) contacting saidsample with a second enzyme that reconverts cystathionine tohomocysteine with the attendant release of by-products; and (c)determining the amount of homocysteine in said sample by measuring theamount of by-products produced by the conversion of cystathionine tohomocysteine or by measuring the amount of substrate consumed, whereinsaid conversion of homocysteine to cystathionine and reconversion ofcystathionine to homocysteine is complete within about 15 minutes. 2.The method of claim 1, said first enzyme being cystathionine β-synthase.3. The method of claim 1, said substrate being L-serine.
 4. The methodof claim 1, said second enzyme being cystathionine β-lyase.
 5. Themethod of claim 1, said by-products being selected from the groupconsisting of ammonia, pyruvate, and combinations thereof.
 6. The methodof claim 1, including the step of repeating steps (a) and (b) aplurality of times.
 7. The method of claim 5, said sample furthercontaining lactate dehydrogenase or a derivative thereof and reducednicotinamide cofactor or derivative thereof, said pyruvate beingmeasured by the oxidation of said reduced nicotinamide cofactor tooxidized nicotinamide cofactor.
 8. The method of claim 7, said amount ofoxidized nicotinamide cofactor being measured by monitoring said sampleat 340 nm.
 9. The method of claim 5, said sample further comprisingpyruvate oxidase, horseradish peroxidase, hydrogen peroxide, and achromogen, said pyruvate being measured based on the intensity of anobserved color in a colorimetric reaction.
 10. The method of claim 5,said ammonia being measured by an ammonia sensor.